Method and compositions for isolating metastatic cancer cells, and use in measuring metastatic potential of a cancer thereof

ABSTRACT

The present invention relates to novel methods and compositions for detection and isolation of cancer cells with metastatic potential. The invention further relates to assays for measuring the metastatic potential of such cancer cells and drug screening assays for the identification of agents having anti-metastatic potential. The present invention further provides methods and compositions for inhibiting the metastatic potential of cancer cells by modulating the activity of serine integral membrane proteases [(SIMP) consisting of seprase and dipedidyl peptidase IV (DPPIV)] expressed on the surface of metastasizing cancer cells.

The invention described in this application was made with funds from theNational Institutes of Health, Grant Number R01CA39077. The UnitedStates Government has certain rights in this invention.

1. INTRODUCTION

The present invention relates to novel methods for detection andisolation of cancer cells with metastatic potential from blood, ascitesand tumor tissue derived from subjects with metastatic cancer. Theinvention further relates to novel compositions for use as cell-adhesionmatrices for detection and isolation of cancer cells and for use asblood filters by subjects having metastatic cancers. The methods andcompositions of the invention may also be used in assays designed formeasuring the metastatic potential of isolated cancer cells and foridentification of agents having anti-metastatic potential. In addition,the invention relates to inhibiting the metastatic potential of cancercells by modulating the activity of serine integral membrane proteasesexpressed on the surface of metastasizing cancer cells. The presentinvention is based on the discovery that carcinoma cells isolated frompatients' blood, ascites or tumor preferentially adhere to, degrade andingest collagenous matrix materials. Further, it was discovered that aserine integral membrane protease (SIMP) consisting of seprase anddipetidyl peptidase IV (DPPIV) subunits are activated on the surface ofmigrating cells.

2. BACKGROUND OF THE INVENTION

Problems associated with cancer cell separation from the or tissue ofpatients with metastatic cancer during traditional bone marrow harvestand leukopheresis procedures have been reported (Campana, D. et al.Detection of Minimal Residual Disease in Acute Leukemia: MethodologicAdvances and Clinical Significance, Blood, 1995 Mar. 15, 85(6): 1416-34;Brugger, W. et al., Mobilization of Tumor Cells and HematopoieticProgenitor Cells into Peripheral Blood of Patients with Solid Tumors,Blood, 83(3): 636-40, 1994). It is estimated that among the order of 10billion total mononuclear cells harvested from a patient, there are 25thousand to 12 million contaminating cancer cells. These contaminatingcancer cells have been shown by genetic marking to contribute to relapse(Rill, E R et al., Direct Demonstration that Autologous Bone MarrowTransplantation for Solid Tumors Can Return a Multiplicity ofTumorigenic Cells, Blood, 84(2): 380-383, 1994).

Large numbers of cancer cells were also found in the circulation ofcancer patients with metastatic diseases. Glaves, D., R P Huben, & L.Weiss (1988. Br. J. Cancer. 57:32-35) took samples of blood from therenal vein in 10 patients just prior to surgery of renal cell carcinomaand estimated that cancer cells were being released at rates of 10⁷ to10⁹ cells per day. How these circulating cancer cells contribute tometastasis remains unknown. A major stumbling block is the difficultyinvolved in identifying an extremely minor subpopulation of circulatingcancer cells, ranging from one of thousands to millions of cells, whichare metastatic. It is apparent that majority of circulating cancer cellsare killed due to host immunity. For examples, in experimental animaltumor models where the use of antibody-based cell separation is morereliable, it has been estimated that about 10 to 100 million tumor cellsare released into the blood during the growth of transplantable B16melanomas and Lewis lung tumors (approximately 20 days), however, thesecells give rise to less than 100 lung metastases per mouse (Glaves, D.,1983, Br. J. Cancer, 48:665-673). Furthermore, in the large number ofexperiments in which tumor cells have been introduced directly into thecirculation of mice or rats it is rare that more than 0.01% of suchcells form tumor nodules. More commonly the efficiency is two or moreorders of magnitude lower. These experimental data suggest that theinitial release of cancer cells from the primary tumor is not thelimiting factor in metastatic development as only a very small fractionof shed cancer cells are viable, invasive, and, therefore, metastatic.It is essential to develop a cell separation and detection systemtargeting on such metastatic cells for the understanding of mechanism ofmetastasis.

Several methods are known for separating cancer cells from blood or bodyfluids. Such methods include, for example, separating cancer cells oneby one by microdissection (Suarez-Quian et al., 1999, Biotechniques,26:328-35; Beltinger and Debatin, 1998, Mol. Pathol 51:233-6) or byantibody-based methods using fluorescence activated cell sorting(Pituch-Noworolska et al., 1998, Int. J. Mol. Med. 1:573-8), separatingcancer cells coated with antibodies on a magnetic material through theuse of a magnetic field (Denis et al., 1997, Int. J. Cancer 74:540-4;Racila et al., 1998, Proc. Natl. Acad Sci USA 95-4589-94), or separatingcirculating cancer cells on density gradients (Sabile et al., 1999, Am.J. Clin. Pathol. 112:171-8). However, such cancer cell separationmethods are dependent on the availability of tumor specific antibodiesor the buoyant density and morphology unique to different cancer cells.Thus, a great need exists for efficient methods for removing cancercells from a hematopoietic cell transplant (Gulati, S C et al. Rationalefor Purging in Autologous Stem Cell Transplantation. Journal ofHematotherapy, 2(4):467-71, 1993).

As demonstrated in early studies, primary cancers begin sheddingneoplastic cells into the circulation at an early stage of metastasesformation (Fidler I J, 1973, European Journal of Cancer 9:223-227;Liotta L A et al., 1974, Cancer Research 34:997-1004). Once shed intothe circulation, cancer cells adhere to the basement membrane underlyingvessel walls and invade adjacent connective tissue leading to formationof micrometastases (Liotta et al., 1991, Cell 64:327-336). It ispostulated that cancer cells present in the invasion front and thoseshed into the circulation are critically involved in the progression ofmetastatic diseases.

The metastatic process is complex, involving escape of a cancer cellfrom the primary tumor, movement to a new location and establishment ofgrowth at the new site. To successfully metastasize, the invasive cancercells must acquire the following metastatic properties: (i) sheddingfrom primary carcinoma, (ii) survival in the circulation and growth onvessel wall, (iii) the ability to invade (adhere to, and subsequentlydegrade and ingest) collagenous matrix, and (iv) extravasation,colonization and cooperation with angiogenesis (Chambers et al., 1998,Cancer & Metastasis Review 17:263-269).

The various steps associated with the process are essentially the samewhether the cell escapes into lymphatic or blood vessels, and theyinvolve an essential cellular property, i.e., cell invasiveness. Cancerinvasiveness requires the adhesion to, and the degradation and ingestionof the extracellular matrix (ECM) by invading cancer cells, accompaniedby translocation or migration of the cells into the ECM. Such cellularactivities occur on membrane protrusions referred to as invadopodia,which exhibit dynamic membrane mobility, ECM adhesion, and degradation.Recent evidence has demonstrated the involvement of serine integralmembrane proteases (SIMP), including dipetidyl peptidase IV (DPPIV)/CD26and seprase, in cell surface proteolysis (Chen, W-T, 1996, EnzymeProtein 49:59-71).

SIMP members are type II transmembrane proteins comprising cytoplasmictails that contain 6 amino acids followed by a 20 (seprase) or 22(DPPIV) amino acid transmembrane domain at the N-terminus and a stretchof 200 amino acids at the C-terminus that constitutes a catalytic regionwith the catalytic serine in a non-classical orientation (Goldstein, L Aet al., 1997, Biochem. Biophys. Acta. 1361:11-19). DPPIV specificallyremoves N-terminal dipeptides from oligo-peptides with either L-proline,L-hydroxyproline, or L-alanine at the penultimate position. Suchpeptides include Neuro-Peptide Y and other peptide hormones (Heins, J etal., 1988, Biochem. Biophys. Acta 954:161-169; Walter, Ret al., 1980,Mol. Cell Biochem. 30:111-126). In addition, a recent report showed thatDPPIV also possesses a seprase-like gelatinase and endopeptidaseactivity, suggesting its involvement in collagen degradation (Bermpohl Fet al., 1998, FEBS Letters 428:152-156). In addition, DPPIV is expressedconstitutively on brush border membranes of intestine and kidneyepithelial cells (Yaron and Naider, F, 1993, Crit. Rev. Biochem. Mol.Biol. 28:31-81; Morimoto C. and Schlossman S F, 1994, Immunologist2:4-7) and transiently expressed on T-cells implicating DPPVI as amarker for T-cell activation (Morimoto C. and Schlossman S F, 1994,Immunologist 2:4-7).

Seprase, originally identified as a 170-kDa membrane-bound gelatinase isexpressed on invadopodia of highly aggressive melanoma LOX (Aoyama A.and Chen, W. T., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:8296-8300;Mueller, S C et al., 1999, J. Biol Chem. 274:24947-24952; Monsky, W L etal., 1994, Cancer Res. 54:5702-5710). The active enzyme was isolatedfrom cell membranes and shed vesicles of LOX cells. Seprase is ahomodimer of 97-kDa subunits (Pineiro-Sanchez, M L et al., 1997, J.Biol. Chem. 272:7595-7601). Analysis of the deduced amino acid sequencederived from a cDNA that encodes the 97-kDa subunit reveals that the97-kDa subunit is homologous to DPPIV, and is essentially identical tofibroblast activation protein α (FAPα) (Goldstein et al., 1997 Biochem.Biophys. Acta. 1361, 11-19; Scanlon, M. J et al., 1994, Proc. Natl.Acad. Sci. U.S.A., 91:5657-5661). FAP α is expressed on reactive stromalfibroblasts of epithelial cancers and in healing wounds but not in adulttissue (Garin-Chesa, P. et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:7235-7239).

In carcinoma tumors, however, FAPα was not found to be expressed incarcinoma or endothelial cells (Garin-Chesa et al., 1990, Proc. Natl.Acad. Sci. 87:7235-7239). Seprase and FAPα differ mainly in a stretch of45 amino acid residues contiguous with the highly conserved motif GXSXGthat contains the active site serine (Scanlan et al., 1994, Proc. Natl.Acad. Sci. USA 91:5657-5661; Goldstein et al., 1997 Biochem. Biophys.Acta. 1361, 11-19). Recently, an alternatively spliced seprase mRNA wasidentified from the human melanoma cell line LOX that encodes a noveltruncated 27-kDa seprase isoform, that precisely overlaps thecarboxyl-terminal catalytic region of 97-kDa seprase (Goldstein andChen, 2000 J. Biol. Chem. 275:2554-2559). The splice variant mRNA isgenerated by an out-of-frame deletion of a 1223-base pair exonic regionthat encodes part of the cytoplasmic tail, transmembrane, and themembrane proximal-central regions of the extracellular domain (Val(5)through Ser(412)) of the seprase 97-kDa subunit. It is possible thatseprase exhibits both gelatinase and Gly-Pro-dipeptidase activities,while the truncated seprase only has the latter dipeptidase activity.

It has long been believed that collagen remodeling is mediated by matrixmetalloproteinases (MMP). However, trials with MMP inhibitors(Marimastat, AG3340) and angiogenic inhibitors (angiostatin andendostatin) in patients with cancer have not produced obvious evidenceof anti-metastatic, anti-invasive effects. These data indicate thatother enzyme systems are needed to replace MMP at the invasion front ofa tumor.

Cancer cell invasiveness in vitro can be a direct indication of atumor's metastatic potential. Knowledge of the cell's invasive phenotypeis important in developing cancer treatments that maximize patientsurvival and quality of life. It is also important in its use informulating diagnostic tools for detecting cancer progression andmetastasis. Therefore, much effort has focused on measuring cancer cellinvasiveness, a characteristic of the metastatic potential ofcarcinomas.

Invasiveness of a cell is often inferred by its cell surface proteolyticactivities that degrade extracellular matrix (ECM) components, and thatinternalize ECM fragments. In Vitro assays for such activities are oftencomplicated by other cell surface phenomena such as adhesion, cellsurface proteolysis, and membrane mobility. One particular assaydesigned to measure invasiveness of a cell involves the covalent linkageof fluorescence-labeled or radio labeled fibronectin (or other ECMcomponents) to the surface of a cross-linked gelatin substrata (Chen etal., 1984, J. Cell Biol 98:1546-1555; Chen, et al., 1985, Nature,316:156-158; Chen et al., 1989, J. Exp. Zool. 251:167-185; Chen et al.,1994, J. Tiss. Cult. Meth. 16:177-181; Meuller et al., 1989, J. Cell.Biol. 109:3455-3464). In this particular technique fibronectin waslabeled and used to coat over-fixed protein film. The film was then usedto measure cell surface proteolytic activities as well as the cellularinvasive phenotype in terms of foci of invadopodial extensions andsurface indentations in the film. However, this fibronectin-gelatin filmassay is of limited value because (i) it uses conventional, over-fixedprotein films; (ii) it lacks sensitivity for detecting proteolyticactivity of moderately invasive cells such as most tumor cell lines inculture, fibroblasts and angiogenic endothelial cells, (iii)cross-linked gelatin fragments are not ingested by cancer cells; and(iv) it is difficult to build a three dimensional culture gel systemfrom fibronectin and cross-linked gelatin materials. Thus, reliableprocedures to measure the invasiveness of such cells will havesignificant impact in both clinical diagnostic and therapeuticapplications of cancer.

The present invention provides a unique, functional based cellseparation method to isolate various forms of cancer cells from blood,ascites and primary tumor tissue of patients with metastases, andperipheral blood mononuclear cells including endothelial cells fromblood of normal donors. Additionally, the present invention providesevidence that seprase and/or DPPIV are selectively induced in invasivecarcinoma cells and in activated fibroblasts (or other tissue cells) andsprouting endothelial cells of malignant tissues thereby providingtargets for development of drugs for inhibiting tumor invasion andmetastasis.

3. SUMMARY OF THE INVENTION

The present invention relates to a novel cancer cell capture system forrapid and efficient detection and selection of invasive cancer cellsfrom the blood, ascites and/or tissue of cancer patients. In later stagecancer patients, some cancer cells were found associated with bloodcomponents to form large aggregates of cells. Such clumping of cells maycontribute to organ dysfunction associated with late stage cancer. Thepresent invention provides compositions and methods that can detect suchcells in the blood of cancer patients with metastatic diseases, and maybe used to remove such aggregates of cells as well as free-livingindividual cancer cells from the patient's blood, ascites and/or tissue.

In contrast to earlier antibody approaches for cancer cell isolation anddetection, the present cell separation system is based on the functionalproperties of the cancer cells, i.e., their ability to adhere to,degrade, and ingest the extracellular matrix. Thus, the cell separationand assay system of the prevent invention is designed to identify andisolate the very small fraction of cancer cells in the blood, ascitesand tumor tissue of cancer patients that are viable, invasive andmetastatic. The enriched cancer cell population can be used, forexample, to determine their metastatic potential and the most effectivetreatment regime. The enriched cells may also be used in fusions withdendritic cells for cancer vaccine development.

The invention relates to natural fibrous compositions comprisingblood-borne adhesion molecule-coated collagen, fibrin, cotton andplastic fibers, to be used as cell-adhesion matrices for use as bloodfilters by subjects having metastatic cancers. Type I collagen derivedfrom, for example, placental tissues or rat-tail, is particularly usefulfor formation of the matrix which can be readily assembled into any formand be coated on vessel surfaces through cycles of polymerization andde-polymerization. The collagen matrix is coated with a variety ofdifferent adhesion molecules derived from blood, including but are notlimited to fibronectin, laminin and vitronectin. Adhesion of individualcancer cells or cell clusters to such a cell-adhesion matrix provides abasis for cellular isolation.

The compositions can be used, for example, to remove undesired cancercells and to enrich hematopoietic progenitor cells from the blood orbone marrow for use as donor cells in bone marrow transplantation.Further, specific cancer cells of different carcinoma cancers may beenriched from whole blood by the cell separation methods of the presentinvention, subjected to ex vivo expansion, then used to interact withdendritic cells to develop an effective tumor vaccine. In addition,circulating cancer cells could be isolated from the patient and thensubjected to a battery of chemotherapeutic regimes in vitro. Effectivedoses or drug combinations could then be administered to that samepatient.

The compositions and methods of the invention provide a rapid method fordetection and isolation of invasive cancer cells in blood, ascites andtissues of patients with cancer. Such cancers include, but are notlimited to, prostate, breast, colon, lung, head & neck, brain, bladder,lymphoma, ovarian, renal & testis, melanoma, liver, pancreatic or othergastrointestinal cancers. Cancer cells are detected and characterizedusing the immunocytochemistry and cellular functional assays of theinvention, i.e., collagen degradation and ingestion.

The method of the invention can be used to develop sensitive assays forthe determination of invasive cancer cells in the blood, ascites ortumor tissue of cancer patients for use in prognostication, monitoringtherapeutic and surgical treatments and early detection of cancer. Thesensitivity and accuracy of measuring the metastatic potential of acancer may be further enhanced using additional assays known to those ofskill in the art, such as determining the tissue origin of cancer cells,measuring the angiogenic capability of the cells, and determiningreduced leukocyte or complement association.

The cell-adhesion matrix of the invention also provides a cancer celltrap that allows for the high yield and efficient removal of viablecancer cells from whole blood, buffy coat, peripheral blood stem cellpreparation or bone marrow. The cell separation method of the inventionis intended for use in therapeutic apheresis and leukopheresis, in whichautologous blood transfusions are done from which contaminating cancercells have been removed. The present invention provides a highlyefficient method for removal of cancer cells from whole blood ofpatients with prostate, breast, colon, lung, head & neck, brain,bladder, lymphoma, ovarian, renal & testis, melanoma, liver, orpancreatic and other gastrointestinal cancers.

Assays are also provided that may be used to screen for agents capableof inhibiting metastasis, thereby modulating the metastatic potential ofcancer cells. Such assays involves contacting the cell-adhesion matrixwith a cancer cell sample in the presence of a test agent and subsequentdetection and quantitation of cancer cell adhesion to and degradation oringestion of the matrix. The assay system of the invention can also beused to monitor the efficacy of potential anti-cancer agents duringtreatment. For example, the metastatic potential of cancer cells inwhole blood can be determined before and during treatment. The efficacyof the agent can be followed by comparing the metastatic potential ofthe cancer cells throughout the treatment. Agents exhibiting efficacyare those which are capable of decreasing cancer invasiveness,increasing host immunity, and suppressing cancer proliferation, buthaving little or no effect on normal tissue cells. For example, such ananti-metastatic drug screening assay system has identified monoclonalantibodies and peptide inhibitors directed against seprase-DPPIV complexthat block the ability of migratory cells to adhere, degrade, and invadea collagenous matrix (FIGS. 13 and 14). Furthermore, such assay systemdemonstrates that anti-sense and ribozyme constructs against seprase andDPPIV are capable of decreasing the invasiveness of breast carcinomacells (FIG. 19).

Methods and compositions are provided for inhibiting the metastaticpotential of cancer cells comprising the administration of modulators ofserine integral membrane proteases. This aspect of the invention isbased on the observation that formation of a novel protease complex,comprising seprase and DPPIV, is a prerequisite for cell invasion intothe collagen matrix. Such protease inhibitors include but are notlimited to those capable of inhibiting the activity of DPPIV andseprase. The discovery that activation of seprase and DPPIV activity isa prerequisite for cell invasion and migration provides target moleculesfor drug screening assays designed to identify inhibitors of cancer cellmigration.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Composition and preparation of a cell-adhesion matrix. FIG.1A. Preparation of the type I collagen based, cell-adhesion matrix,illustrating seven steps involved in matrix preparation, cellseparation, and microscopic measurement of the invasiveness of a cancercell. FIG. 1B. Utilization of the type I collagen based, cell-adhesionmatrix in microtiter measurement of overall collagen degradation by thecells. The degradation and ingestion of the Bodipy-rhodamine collagenmatrix by cancer cells are measured with a fluorescent microtiter platereader as an increase in red fluorescence. The bottom graph showsrelease fluorescence unit (RFU) is proportional to the amount ofBodipy-rhodamine collagen overlaid on each microtiter well in thepresence of bacterial collagenase at 37° C. for 30 minutes.

FIGS. 2A-B. Diagnostic and therapeutic applications of the cell-adhesionmatrix based, cell separation and assay system. FIG. 2A. Cross-sectionalviews of cell separation and assay methods that may be used fordiagnostic applications, including diagnostics, monitoring therapeuticor surgical responses, and early cancer detection. The steps ofisolating, detecting and characterizing cancer cells from blood, ascitesor tissue cell populations is illustrated. FIG. 2B. Cross-sectionalviews of cell separation and assay methods of the present invention thatmay be used for therapeutic and preventive applications. The steps ofisolating and culturing cancer cells from blood, ascites or tissue cellpopulations are illustrated.

FIGS. 3A-F. Cancer cell separation analyses of blood samples frompatients and controls. Representative examples of fluorescent-taggedHs578T breast carcinoma cells spiked in complete medium alone, or theblood of control normal donor and a bladder cancer (BLC) patient areshown. Red fluorescent-tagged Hs578T breast carcinoma cells were addedto 3-ml of medium or blood samples, the cells were captured by acollagenous matrix in a microtiter well and cultured for 18 hours. Theimages shown were taken with super-imposed illumination of fluorescenceand phase contrast, and represent only a portion of the microtiter well.FIG. 3A. Fluorescent analysis of Hs578T breast carcinoma cells (500cells) added to the medium alone. Estimated 495 cells were found asbright spots attached and spread on the matrix. FIG. 3B. Fluorescentanalysis of Hs578T breast carcinoma cells (500 cells) spiked in theblood of a normal donor. Estimated 415 red fluorescent carcinoma cellswere found among the other blood cells (without fluorescent signal) inthe culture that remained on the matrix substratum. FIG. 3C. Fluorescentanalysis of Hs578T breast carcinoma cells (19 cells) spiked in the bloodof a normal donor. Estimated 11 red fluorescent carcinoma cells werefound among the other blood cells (without fluorescent signal) in theculture that remained on the matrix substratum. FIG. 3D. Fluorescentimage of Hs578T breast carcinoma cells (200 cells) spiked in the bloodof a BLC patient. Estimated 182 Hs578T breast carcinoma cells along withcirculating BLC cells were captured from blood of cancer patient. Hs578Tcells tend to form aggregates with mononuclear blood cells of thepatient seen as dark spots in this figure. FIG. 3E. Fluorescent analysisof matrix-captured Hs578T breast carcinoma cells (19 cells) spiked inthe blood of a BLC patient. Estimated 16 red fluorescent carcinoma cellswere found among the other blood cells and one Hs578T breast carcinomacell is seen in the field. FIG. 3F. Immunofluorescent analysis ofmatrix-captured Hs578T breast carcinoma cell shown in FIG. 3E andcirculating BLC cells. Here, the Hs578T breast carcinoma cell and fourbladder carcinoma cells derived from the blood were stained withfluorescein-conjugated anti-cytokeratins PCK antibody. The Hs578T breastcarcinoma cell (shown as red fluorescence in FIG. 4E) can be seen amongthe green fluorescent cytokeratin-labeled bladder carcinoma cellsderived from patient's blood. Picture size A-D, 662 μm×478 μm; Picturesize E-F, 331 μm×239 μm.

FIGS. 4A-I. Circulating cells attached to cell-adhesion matrix. FIGS.4A-C. Circulating cells aggregated on a piece of tissue fragment inblood of a patient with squamous cell carcinoma of head and neck (HN).The cell-tissue cluster was captured on the substratum using acollagenous matrix, and cultured for four days (d4 in A), six days (d6in B), and 13 days (d13 in C). FIG. 4D. Cells aggregated on a piece oftissue fragment in blood of a patient with metastatic prostate cancer(PC), and cultured for three days (d3). FIG. 4E. Circulating carcinomacells from a patient with metastatic prostate cancer (PC) aggregated ona fibrin fiber (fibrin), and cultured for three days (d3). FIG. 4F.Circulating cells from a patient with metastatic prostate cancer (PC)aggregated on plastic scraps (plastic), and cultured for three days(d3). Putative cancer cells of large size can be seen to preferentiallyadhere on plastic scraps in the field. FIG. 4G. Circulating cells in thesame culture as in F, but cultured for twelve days (d12). Putativecancer cells of large size can be seen to preferentially adhere onplastic scraps in the field. FIG. 4H. Circulating cells from a patientwith metastatic prostate cancer (PC) aggregated on purified cotton(cotton), and cultured for three days (d3). FIG. 4I. Growth of putativecancer cells from a patient with brain cancer (BN) aggregated onpurified cotton (cotton), and cultured for 20 days (d20). Picture sizeA-G, 662 μm×478 μm; Picture size H-I, 1324 μm×956 μm.

FIGS. 5A-H. Circulating cells attached to type I collagen-based,cell-adhesion matrix. FIGS. 5A-C. Cell aggregates from a patient withmetastatic pancreatic cancer (PnC) captured on the substratum using thetype I collagen matrix, washed, and cultured for five days (A), ninedays (B), and 16 days. (C) on the matrix. These illustratephase-microscopic appearance of a type I collagen based, cell-adhesionmatrix and carcinoma cells, that invade the matrix and grow on theplastic surface. Carcinoma cells, not present in blood of most normaldonors, have distinct morphology and increase their cellular size andnumber in culture, while co-isolated leukocytes are small and reducetheir number in culture. FIG. 5D. Circulating cell aggregates from apatient with metastatic pancreatic cancer (PnC) captured on thesubstratum using the type I collagen matrix. Cells were eluted from thematrix and cultured in complete medium for seven days on plastic surface(plastic). Spindle-shaped cells associate with epithelial-shaped,carcinoma cells at the periphery of a cellular colony. FIG. 5E-F.Invasion of stromal fibroblasts into a type I collagen substratum. Cellswere dissociated and isolated from a tumor biopsy of a patient withsquamous cell carcinoma of head and neck (HN). Two morphologicallydistinct, cell types were isolated on the surface of the matrix: HNsquamous cells remain quiescent on the matrix surface (E), while stromalfibroblasts invade into the collagen gel (F), approximately 80 μm belowthe level seen in FIG. 5E as measured by the fine stage adjustment dialof Nikon Eclipse TE300 inverted microscope. FIG. 5G. Morphology of atype I collagen based, cell-adhesion matrix illustrating transmissionelectron-microscopic appearance of the matrix and its adherent cancercells, that were isolated from the blood of a patient with metastaticcolon cancer (CC). FIG. 5H. A high magnification view of the collagenfibers shown in FIG. 5H, illustrating transmission electron-microscopicappearance of the assembled collagen fibers. Picture size A-F, 662μm×478 μm; Picture size G-H, indicated in the bar area.

FIG. 6A-D. Morphological basis of the invasive phenotype of circulatingcarcinoma cells. FIGS. 6A-B. Circulating cell aggregates from a patientwith metastatic colon cancer (CC). Cells were freshly isolated andcultured for one day (d1) and nine days (d9). These illustratephase-microscopic appearance of degraded holes in the type I collagenbased, cell-adhesion matrix generated by carcinoma cells andcharacteristics of carcinoma cells, that have distinct morphologies,i.e., small in size on d1 (A) but large and epithelial shape on d9 (B).Carcinoma cells increase their cellular size and number in culture,while co-isolated leukocytes are small and reduce their number. Also,spindle-shaped cells associate with epithelial-shaped, carcinoma cellsin colonies (B). FIGS. 6C-D. Circulating cell aggregates from a patientwith metastatic stomach cancer (SC). Cells were captured on thesubstratum using the type I collagen matrix, and cultured for 19 days.Epithelial-shaped, carcinoma cells grow on plastic surface in degradedholes of collagenous film (C) or line the edge of collagen film (D).Picture size A-B, 1324 μm×956 μm; Picture size C-D, 662 μm×478 μm.

FIGS. 7A-D. Cells from control blood samples that are isolated by thecell separation method. FIG. 7A. Cells isolated from the blood of a 39year-old normal donor and their underlying type I collagen matrix. Veryfew cells survived after 7 days in culture, while the matrix membranestayed intact. FIG. 7B. Cells isolated from the blood of a patient withbenign colon tumor (CTN) and their underlying type I collagen matrix.Very few cells were isolated, and the matrix membrane stayed intact.FIGS. 7C-D. Circulating cells of a patient with metastatic breast cancer(BC) and their underlying type I collagen matrix. Blood sample wascollected during treatment with chemotherapeutic compounds. Relativelyfew cells were isolated and survived in d1 (C) and d4 (D) cultures, andthe matrix membrane stayed intact. Picture size A-D, 662 μm×478 μm.

FIGS. 8A-L. The cell separation and assay system for the cell invasivephenotype using fluorescently labeled collagen, and immunocytochemistryof circulating carcinoma cells using antibodies against epithelialmarkers. Cells were derived from the blood of patients with breastcarcinoma (BC, A-C), squamous cell carcinoma of head-neck (HN, D-F),colon cancer (CC, G-I), prostate cancer (PC, K), and a normal donor(Control, L), as well as the Hs578T breast carcinoma cell line (Hs578T,J). Cells were captured on the substratum using a rhodamine-collagenmatrix, and cultured for one day (d1) or 63 days (d63). FIG. 8A-C.Ingested rhodamine-collagen blotch as a marker for the invasivephenotype of circulating carcinoma cells in a patient with breast cancer(BC). Cells were seeded on the matrix for 18 hours and fixed forexamination. Among numerous leukocytes in the background, individual andcluster of putative cancer cells (PH, phase contrast image shown in A)were shown to ingest rhodamine-collagen fragments, marked Col+ (redfluorescence shown in B). Super-imposed image shown in panel Cdemonstrates the exact match of rhodamine-collagen spots to putativecancer cells. These represent the enumeration of 2,067 Col⁺ cells per mlof patient's blood. FIGS. 8D-F. Ingested rhodamine-collagen blotch as amarker for the invasive phenotype of circulating carcinoma cells in apatient with squamous cell carcinoma of head-neck (HN). A large clusterand individuals of putative cancer cells (PH, phase contrast image in D)were shown to be coincident with cells positively antibody-stained forbroad-spectrum cytokeratin subunits (PCK⁺, in E) and these loaded withrhodamine-collagen Col+ (red fluorescence in F). These represent theenumeration of 20,814 PCK⁺ and 18,003 Col⁺ cells, respectively, per c.c.of patient's blood. The high number of PCK⁺ compared to these of Col⁺cells may be due to the presence of less viable cancer cells that arepositive for antibody staining, and that adhere to the cell-adhesionmatrix. FIGS. 8G-J. Ingested rhodamine-collagen blotch as a marker forthe invasive phenotype of circulating carcinoma cells in a patient withcolon cancer (CC). Clusters and individuals of putative cancer cells(differential interference contrast image in G) were labeled withantibodies against epithelial surface antigen (ESA⁺; stained red in G).ESA⁺ cells were shown to be coincident with these stained withantibodies against the endothelial marker factor VIII (F8⁺; greenfluorescent cells in H) and these ingested rhodamine-collagen fragmentCol⁺ (red fluorescent cells in I). These represent the enumeration of2,284 ESA⁺, 7,308 F8⁺, and 1,978 Col⁺ cells, respectively, per ml ofpatient's blood. The high number of F8⁺ compared to these of Col⁺ orESA⁺ cells may be due to the presence of normal endothelial cells in theblood. FIG. 8J. The Hs578T breast carcinoma cell line stained withanti-PCK antibodies, as carcinoma (epithelial) marker control. FIG. 8K.PC cells in culture for 63 days (d63) stained ESA⁺. Note that theselarge cells contain multiple nuclei and increase their diameter over5-folds as compared to d1 cells in G. FIG. 8L. Rhodamine-collagen matrixthat remains intact after culturing blood cells from a normal donor forone day (d1). Picture size A-C, 1324 μm×956 μm; Picture size D-F, 331μm×239 μm; Picture size G-L, 662 μm×478 μm.

FIGS. 9A-L. Angiogenic propensity of circulating carcinoma cells. Cellswere derived from the blood of patients with squamous cell carcinoma ofhead-neck (HN, A-C; I-J), colon cancer (CC, D-F) and prostate cancer(PC, G-H). Cells were captured on the substratum using the collagenmatrix, and cultured for 1-11 days as indicated by d1 or d11. FIGS.9A-C. A large cluster and seven individual HN cells (phase contrastimage shown in A) that were labeled strongly for F8⁺ (B) and thatingested collagen fragment Col+ (C). The HN cells shown in phasecontrast (PH) image (A) are closely associated with these in the samefield that exhibited positive reactivity with F8 antibodies (B) andthese ingested rhodamine-collagen matrix (C). These represent theenumeration of 42,495 F8⁺ and 15,611 Col⁺ cells, respectively, per c.c.of patient's blood. The high number of F8⁺ as compared to Col⁺ cells isdue to the presence of F8⁺ endothelial cells in the blood. FIG. 9D-F. Asubset of CD31⁺ and F8⁺ cells representing the Col⁺ CC cells. The cellson the rhodamine-collagen matrix were labeled with antibodies againstthe endothelial cell marker CD31 and fluorescein conjugated antibodiesagainst the endothelial marker factor VIII (F8). The association ofbright red stained CD31⁺ cells with CC cells was shown in differentialinterference contrast (DIC) image (D). CC cells in the same fieldexhibited positive reactivity with F8 antibodies (E) and these ingestedrhodamine-collagen matrix (F). These represent the enumeration of 11,693CD31⁺, 6,577 F8⁺ and 2,558 Col⁺ cells, respectively, per c.c. ofpatient's blood. The high number of CD31⁺ and F8⁺ as compared to Col⁺cells is due to the presence of CD31⁺ and F8⁺ endothelial cells in theblood. FIGS. 9G-H. PC cells incorporating acetylated LDL. The bright redstained epithelial surface antigen (ESA) positive cells shown indifferential interference contrast (DIC) image (G) display the ingestionof the fluorescein-LDL (H). These represent the enumeration of 9,744ESA⁺ and 34,105 LDL⁺ cells, respectively, per c.c. of patient's blood.The high number of LDL⁺ as compared to ESA⁺ cells is due to the presenceof other endothelial cells that incorporate acetylated LDL. FIGS. 9I-J.Primary cultured HN cells incorporating acetylated LDL. Note that onlyone specific cell toward the center incorporate both fluorescein-LDL andrhodamine-collagen as shown in the triple fluorescein-Rhodymenia-Hoechstimage (J), suggesting that this cell represents a cell retaining theinvasive phenotype after in culture for 12 days. FIGS. 9K-L. Capillarynetwork development by circulating CC cells on collagen gel. Networkformation (K) and cord-like structures (L) were observed 2 days afterplating circulating CC cells on type I collagen gel, 0.5-mm inthickness. Picture size A-C; G-J, 331 μm×239 μm; Picture size D-F; K-L,662 μm×478 μm.

FIGS. 10A-L. Immune cancer killing and growth of circulating cancercells in culture. Cells were isolated from the blood of patients withcolon (CC, A-E), prostate (PC, F-I), or bladder cancer (BLC, J-L) usingthe collagen matrix, and cultured in the medium containing 10-20% humanplasma. FIG. 10A-C. CD34⁺ peripheral blood stem cells clustering withthe F8⁺/Col⁺ CC cells. The association of bright red stained CD34⁺ cellswith CC cells was shown in the differential interference contrast (DIC)image (A). CC cells in the same field exhibited positive reactivity withF8 antibodies (B) and these ingested rhodamine-collagen (C). Theserepresent the enumeration of 111,082 CD34⁺, 7,673 F8⁺ and 2,558 Col⁺cells, respectively, per c.c. of patient's blood. The high number ofCD34⁺ and F8⁺ as compared to Col⁺ cells is due to the presence of CD34⁺stem cells and F8⁺ endothelial cells in the blood. FIGS. 10D-E. CD45⁺leukocytes clustering with the Col⁺ CC cells. The isolated cells on therhodamine-collagen matrix were labeled with antibodies against theleukocyte common antigens CD45. The association of bright red stainedCD45⁺ leukocytes with CC cells was shown in the DIC image (D). CC cellsin the same field exhibited the ingestion of the rhodamine-collagenousmatrix (E). These represent the enumeration of 125,670 CD45⁺ leukocytesand 1,827 Col⁺ cells, respectively, per c.c. of patient's blood. FIGS.10F-I. Sequential views of cytolysis of immune and cancer cell clustersfrom a patient with prostate cancer (PC) who exhibited favorableprognosis. Most cancer cells were attacked by leukocytes and becamefragmented after one day in culture (F-H). However, leukocytesdisappeared after seven days in such culture, leaving behind a few PCcolonies (1). FIGS. 10J-L. Cytolysis of bladder cancer (BLC) cells dueto autoimmune plasma derived from the same patient. BLC cells werecultured in the presence of 10% autologous plasma, derived from theblood of the same cancer patient (au.plasma in J-K), or those from anormal donor (n. plasma in L). BLC cells became lysed and brown in colorin the medium containing autologous plasma in the second day (au.plasmain J-K), while they stayed viable for over 6 weeks in the mediumcontaining normal plasma (n.plasma in L), suggesting a role ofauto-cancer antibodies in complement cytolysis and signifying the hostimmunity against metastasis. Picture size A-L, 662 μm×478 μm.

FIGS. 11A-B. Gel Filtration Column Chromatography and Immunoblotting forSepharase and DPPIV. FIG. 11A. WGA-purified, detergent-soluble proteinsderived from WI38 cells were separated by a gel filtration column ofSepharase 12 (Pharmacal-LKB, Piscataway, N.J.). Protein standards usedto calibrate the column were vitamin B-12 (1.35-kDa), myoglobin(17-kDa), ovalbumin (44-kDa), gamma globulin (158-kDa), catalase(232-kDa), ferritin (440-kDa), and thyroglobulin (670-kDa). FIG. 11B.Fractions were analyzed by immuno-dot blotting using mAbs againstseprase and DPPIV. Seprase and DPPIV were found in 200-kDa (Fraction17), 440-kDa (Fraction 14), and 670-kDa (Fraction 13) ranges, suggestingthe presence of the seprase-DPPIV complex at 440-670 kDa sizes.

FIGS. 12A-D. Characterization of the Seprase-DPPIV Complex Derived fromWI38 Human Embryonic Lung Fibroblasts. FIG. 12A. Immunoprecipitation(Ip) of surface-biotinylated WI38 fibroblasts. Both mAbs; againstseprase (D28) and DPPIV (E19) identified a RIPA-solubilized proteincomplex that contains two major bands at 170- and 200-kDa, indicative ofseprase and DPPIV, respectively. FIG. 12B. The seprase-DPPIV complexdemonstrated by Ip and immunoblotting (Ib). Seprase- orDPPIV-immunoprecipitates isolated from WI38 RIPA lysates were confirmedin seprase- or DPPIV-immunoblots but not in β1 or β3 integrin blots,suggesting that the protease complex is not associated with β1 and β3integrins in RIPA. FIG. 12C. Gelatinolytic activity of the seprase-DPPIVcomplex. The protease complexes were analyzed by gelatin zymography inthe absence of Ca⁺⁺ and in the presence of 2 mM EDTA. Both seprase andDPPIV immuno-isolates (Ip) exhibit a 170-kDa gelatinase (seprase)activity. FIG. 12D. DPPIV proline-specific peptidase activity of thecomplex. Both seprase and DPPIV immuno-isolates (Ip) exhibit samepeptidase activity using fluorescent Ala-Pro-AFC(7-Amino-4-Trifluoromethyl Coumarin) substrate overlay assay. Noactivity could be observed for αv, α2, α6 or β3 integrin or controlimmuno-isolates.

FIGS. 13A-E. Cell Migration in Collagen Gel and Collagen Degradation byWound-Activated WI38 Fibroblasts. FIG. 13A. Morphology of WI38 at 1 h(a, b, c) and 18 h (d, e, f) after wounding of the cell monolayer(photographed while cells were alive). Panel a & d: phase contrast ofWI38 at the interface between the wound edge and cell-free glasssurface, showing that spindle-shaped cells migrated on collagen fibersat 18 h (d). Panel b & e: fluorescent collagen gels in same fields shownin panels a & d. Uniform layer of TRITC-labeled collagen is seen at 1 h(b) but local removal of fluorescent collagen occurred at the wound edgeby activated migrating cells at 18 h (e). Panels c & f: microscopicsuperimposed image of left and middle panels. Bar=10 μm. FIG. 13B.Dose-dependent inhibition of cell migration by inhibitory mAb E19(against DPPIV) but not by control mAb C37 (against a cell surfaceglycoprotein gp-90). Three experiments of 24 h monolayer wound modelswere used for each antibody. Cell migration was quantified by measuringthe areas of cell advancement from the original wound edge. The valuesare mean±SD. FIG. 13C. Reversal of antibody inhibition of cellmigration. All antibodies, mAb E19 (against DPPIV) and mAb C37 (againstgp-90), were applied at 5 μg per ml. After day 3, antibodies wereremoved and the antibody inhibitory effect was reversed. FIG. 13D.Histograms of the local removal of fluorescent collagen gel bywound-activated cells. Collagen degradation was measured as areas offluorescent collagen removal by migratory cells. mAb E19 (against DPPIV)inhibited collagen removal by migratory cells while control mAb C37(anti-gp-90) did not. All antibodies were applied at 5 g per ml. Threeexperiments were used for each antibody. The values are mean±SD. FIG.13E. Collagen degradation by migratory cells in a sparse culture.Collagen degradation was measured by the release of fluorescent collagenpeptides from a collagen gel by migratory WI38 cells. Bacterialcollagenase was used as a positive control for fluorescent peptiderelease. All antibodies were applied at 5 μg per ml. Three experimentswere used for each antibody. The values are mean±SD.

FIGS. 14A-B. Attachment and Spreading of WI38 Cells on CollagenSubstratum are Mediated Primarily by β1 Integrins but Not by DPPIV. FIG.14A. Inhibition of WI38 cell spreading on collagen substratum by mAb C27(against β1 integrins) but not by mAb E19 (against DPPIV) or mAb C37(anti-gp-90). FIG. 14B. Inhibition of WI38 cell attachment to collagensubstratum by mAb C27 (against β1 integrins) but not by mAb E19 (againstDPPIV) or mAb C37 (anti-gp-90). Each value represents the mean of threeseparate determinations ±S.D. Duplicate experiments gave similarresults.

FIG. 15. Co-localization of Seprase and DPPIV at Invadopodia of WI38Cells Migrating in Collagen Gels. Phase contrast image of invadopodia(indicated by open arrow) in a WI38 cell migrating in type I collagengel (a). Immunofluorescent image of DPPIV in the invadopodia (indicatedby open arrow) that was labeled directly with TRITC-mAb E19 againstDPPIV(b). Immuno-fluorescence image of seprase in the invadopodia(indicated by open arrow) that was labeled directly with FITC-mAb D28against seprase (c). Superimposed image of panels b and c, showing thatseprase and DPPIV co-localized at the invadopodia (indicated by openarrow) of a WI38 fibroblast migrating in collagen gel (d). Bar=10 μm.

FIG. 16. Seprase and DPPIV Distribution in Connective Tissue Cells ofHuman Malignant Breast Carcinoma as demonstrated by immunohistochemistry of serial sections of paraffin-embedded tumor tissue. Bothseprase and DPPIV are present in fibroblast-like cells of connectivetissue immediately adjacent to invasive breast carcinoma but not in thatof distant normal tissues (not shown). Arrows indicate positive brown,seprase or DPPIV stains for fibroblasts. Clusters of carcinoma cells arealso positive for seprase (large cell aggregate in middle and bottompanels) and for DPPIV (large cell aggregate in top panel). Paraffinsections of breast carcinoma and adjacent normal tissue were placed inthe same slide and stained with mAb D8 against seprase (middle andbottom panels) or mAb E26 against DPPIV (top panel). Bar=100 μm.

FIG. 17. Distribution of Seprase and DPPIV in Mucosa Cells of HealingHuman Gingival Wounds. Frozen sections of healing mucosa wounds at3-day-old (a, b, c, d and g) and at 7-day-old (e, f and h) were stainedwith hematoxylin and eosin (a and e), with TRITC-mAb D28 against seprase(b-d, and f) or with mAb E19 against DPPIV followed by TRITC anti-ratsecondary antibody (g and h). In 3-day-old wound (a-d, and g),connective tissue (CT) contained cells that were strongly stained withseprase (b, c, and d) and DPPIV (g) antibodies. A confocal microscopeimage shows seprase localization at invadopodia in the form of filopodia(arrows) and cell bodies of fibroblast-like cells in connective tissue(panel d). In 7-day-old wounds (e, f and h), seprase staining was found(f) in the wound granulation tissue (GT), while DPPIV did not (h).Dotted lines indicate the border between granulation and connectivetissues. Letter E indicates wound epithelium; CT, connective tissue; FC,fibrin clot; and GT, granulation tissue. Bar=200 μm.

FIG. 18. Co-localization of seprase and DPPIV in microvessel endothelialcells, fibroblasts, and carcinoma cells at the invasion front of humanmalignant breast ductal carcinoma. Formaldehyde-fixed, paraffin embeddedmalignant human breast ductal carcinoma samples were made in serialsections, seprase distribution was shown by brown staining using mAb D8(left three panels) and DPPIV distribution on an adjacent section usingmAb E26 (right three panels). Top two panels show normal breast tissuesapproximately 2 cm distance from tumor sites. Negative seprase and DPPIVstains were found in epithelial cells (black open arrows), fibrocytes(black arrows) and endothelial cells (black open arrowheads). Middle twopanels indicate infiltrating sheets of poorly differentiated(high-grade) carcinoma cells with predominant brown cellular stains ofseprase and DPPIV in tumor cells (orange open arrows), in fibroblasts(orange arrows), and in some endothelial cells (orange open arrowheads)but not in some larger vessel lining cells (black open arrows). Arrowsin top and middle panels indicate a scale of 100 μm. Bottom two panelsshow low-magnification view of infiltrating sheets of poorlydifferentiated (high-grade) tumor cells with predominant brown cellularstains of seprase and DPPIV in tumor cells (orange open arrows), infibroblasts (orange arrows), and in some endothelial cells (orange openarrowheads). Seprase and DPPIV are specifically expressed in tumor cellsat the invasion front as indicated by most tumor cells in the field butare absent in these in the center of tumors as indicated in the centerof the field. Arrows in bottom panels indicate a scale of 800 μm.

FIG. 19. Relationship of protease expression and collagen-degradingability of MDA-MB-436 cells. A, Release of fluorescent collagen peptidesby MDA-MB-436 cells differentially expressing seprase and DPPIV.Collagen degradation was measured by the release of fluorescent collagenpeptides by parental cells (Parent), cells transfected with vector(pA11), cells transfected with seprase cDNA (pA15) and cells transfectedwith DPPIV ribozyme (pZ8). Three experiments were used for each and thevalues were mean±SD. Star “*” indicates (ρ<0.05 for pA15 seprase senseand pZ8 DPPIV ribozyme transfectants as compared to parent and pA11vector transfected cells. B, Differential expression of seprase andDPPIV in above-mentioned MDA-MB-436 cells. Cells were extracted withRIPA buffer and immunoblots were done using mAb D8 (Anti-seprase) andmAb F4 (Anti-DPPIV).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel method for forming acell-adhesion matrix for isolation and detection of a subpopulation ofcancer cells which are viable, invasive, and metastatic from a samplederived from a subject possessing a cancer. In particular, a method isdescribed that utilizes a collagenous matrix system for isolation anddetection of cancer cells. The invention described in the subsectionsbelow further relates to a novel highly sensitive in vitro assay formeasuring the metastatic potential of a cancer. An important feature ofthe assay includes applicability to cancer cells in blood, bone marrow,ascites, body fluid, and tumor tissue, or any established cancer cellline.

The “cell-adhesion matrix” of the invention provides a cancer cell trapthat allows for the high yield and efficient removal of viable cancercells from whole blood, buffy coat, peripheral blood stem cellpreparation or bone marrow. The cell separation method of the presentinvention may be used for cancer diagnostic purposes, e.g. earlydetection, monitoring therapeutic and surgical responses, andprognostication of cancer progression. The cell separation method of thepresent invention may also be used for cancer preventive and therapeuticpurposes, including the use in blood filter as a cancer cell trap, ingenetic and cytogenetic analyses, in new drug target discovery, and incancer vaccine development.

The in vitro assay system of the invention further provides a screeningmethod for identifying agents with anti-metastatic activity. Such agentsmay be used to inhibit metastatic spread of cancer cells in subjectshaving cancer. In addition, the assay may be used to screen for nucleicacid molecules with anti-metastatic potential. For example, antisenseand ribozyme molecules may be screened for their ability to inhibitmetastasis. In yet another embodiment of the invention, the activity ofseprase or DPPIV may be measured to determine the metastatic potentialof cancer cells.

5.1 The Matrices of the Invention

The present invention provides a novel, natural, cell-adhesion matrixsystem that may be used for the isolation and detection of cancer cellsfrom samples derived from cancer subjects. The natural cell-adhesionmatrix of the invention has specific binding affinity for blood-bornecell adhesion components, including but not limited to fibronectin,laminin and vitronectin. The present invention is based on theobservation that cancer cells present in the circulation of patientswith metastatic diseases can attach to tissue fragments and form largecellular clusters indicating that natural structural scaffolds have ahigh affinity for blood-borne adhesion molecules and, therefore, promoteattachment of metastasized cancer cells. Additionally, it was discoveredthat natural fibers, including type I/III collagen, fibrin, purifiedcotton, and mechanically scratched surfaces of tissue culture plastic,absorb preferentially blood-borne adhesion components that promoteadhesion of cancer cells.

The present invention encompasses cell-adhesion matrices comprisingcollagenous fibers, fibrin gels, purified cotton or plastic fibers foruse as a cellular substratum for the detection and isolation of viablehuman cells in vivo, these cells include but not limited to carcinoma,endothelial and tissue cells. A variety of commercially availablecollagenous materials may be used to prepare the collagen matrix,including, but not limited to, human placental type I collagen,purchased from Calbiochem-Novabiochem Co. (La Jolla, Calif.); andrat-tail type I collagen, available from Collaborative BiomedicalProducts (Becton and Dickinson Labware, Bedford, Mass.). Any form ofrecombinant collagen may also be employed, as may be obtained from acollagen expressing recombinant host cell, including bacterial, yeast ormammalian cells.

Type I collagen can be readily assembled and reassembled into any formand be coated on vessel surfaces through cycles of polymerization andde-polymerization. For example, it is well-known that type I collagenmonomers, at a concentration of over 0.3 mg/ml, polymerize underconditions of natural pH, warm temperatures (25-37° C.) and mediumcontaining saline, and that collagen fibers de-polymerize in thecondition of pH 2-4, cold temperatures (2-10° C.), and low salt medium(Klasson, S. C., et al. 1986, Coll. Relat. Res. 6:397). Such acell-adhesion matrix can be formed lining the wall of a micro capillary,cell isolation columns, tissue culture plates, or micromesh for captureof cancer cells in biological fluids.

The matrix scaffold of the present invention may be composed of naturalfibers, including, but not limited to, collagens, fibrin and purifiedcotton. A common property of these matrix scaffolds is that theirsurfaces have affinity for blood-borne adhesion molecules, including,but not limited to, fibronectin, vitronectin and laminin. The matrixscaffold, when coated with cell adhesion molecules present in wholeblood, plasma or serum, provides an adhesive surface which supports theattachment of cancer cells and tissue cells from high-density cellpopulations. These populations may be derived from blood, lymph, bonemarrow and tumor tissue and may include many different cell types.

The method of preparing a collagenous matrix is described in detail inthis application. A matrix scaffold composed of fibrin fibers wereprepared on vessel's surfaces by reducing the anti-coagulant content ofplasma. Animal or human plasmas were initially diluted to 10-20% withDulbecco's modified Eagle's medium (DMEM) and added into cell isolationvessels or wells. The vessels or wells were incubated for 30 min at 37°C. in a CO₂ incubator to allow polymerization of the fibrin fibers onvessel's surface. Purified cotton fibers were simply suspended in DMEMcontaining 10-20% bovine serum and seeded in cell culture wells forcoating of cell adhesion molecules. Similarly, glass and plastic fiberswere coated with cell adhesion molecules by suspension in DMEMcontaining 10-20% bovine serum. A cell culture plate was scrapped withan Eppendorf peptide tip to prepare as a matrix scaffold. It should benoted that pre-coating of cell adhesion molecules to a matrix scaffoldis not necessary when whole blood or a buffy coat will be applied to amatrix scaffold. However, whole blood or buffy coat should be treatedwith anticoagulants to prevent coagulation during the cell separationprocedure. Specifically, blood or buffy coat were pre-diluted with equalvolume of medium containing 0.5 mM EDTA or with 10% anticoagulantcitrate dextrose (ACD; Baxter Healthcare Corporation, Ill.) containing50 unit heparin/ml.

To facilitate detection of cancer cells bound to the collagenous matrixand to measure the metastatic potential of such cells, the matrix can belabeled with a variety of different agents, including, but not limitedto, fluorescent dyes, biotin, color dyes, and radioactive probes. Forexample, collagen fibers can be labeling by direct conjugation of dyesthereby protecting polymerization sites in the collagen from labeling.Collagen fibers, either labeled by this method or unlabeled, can bereadily disassembled into soluble collagen monomers, which can besubsequently assembled into any form on vessel surfaces. Preferred dyesfor labeling of collagen include Bodipy-rhodamine or fluorescein dyes,available from Molecular Probes, Inc., which are quench-fluorescentdyes. As illustrated in FIGS. 1A and 1B, Bodipy-rhodamine's fluorescentsignal (yellow) is reduced when two dye molecules are situated closetogether on collagen but is increased (red) when collagen is degraded orcleaved by enzymes to separate two dye molecules. Methods for labelingof the matrix are well known to those of skill in the art. Likewise,conjugation of a cytotoxic compound to such collagen fiber can provide anovel vehicle for delivery of the drug to specific cancer cells. Forexample, injection of a cytotoxin-conjugated, collagen microsphere intoa patient with cancer can achieve specific killing of cancer cells.

The surface of a matrix scaffold is further coated with blood-borneadhesion molecules. A variety of commercially available solutionscontaining blood-borne adhesion molecules can be used to coat thematrix, including, but not limited to, calf serum, fetal calf serum(Collaborative Research, Inc., Bedford, Mass.); human serum (Sigma); andhuman plasma fibronectin, laminin and vitronectin (CollaborativeResearch, Inc., Bedford, Mass.). The surface of the matrix scaffold canbe coated with adhesion molecules using either 10-20% of calf serum,fetal calf serum, or human serum (or plasma), or 0.01-0.5 milligram permilliliter of human plasma fibronectin, laminin and vitronectin.

The present invention achieves a highly desirable objective, namelyproviding a method for prevention and intervention of metastasesformation in a cancer subject by a cell separation system. Specifically,the invention encompasses a method for isolation of cancer cells derivedfrom a cancer subject comprising:

-   -   (a) inoculating a cancer cell sample derived from a cancer        subject onto cell-adhesion matrix;    -   (b) incubation of the cancer cell sample for a time sufficient        to allow adhesion of cancer cells to the matrix, followed by        removal of non-adherent cells; and    -   (c) ex vivo propagation of cancer cells bound to the matrix.

The cell separation method of the present invention may be used toisolate any desired cancer cell population from in vivo or in vitrosources including, but not limited to, body fluids, e.g. circulatingblood, urine, bone marrow, spinal and pleural fluids, ascites, sputum;dissociated tumor tissue specimens; and cultured tumor cells. Examplesof desired cancer cell populations include, but are not limited to,carcinoma cells of prostate, breast, colon, brain, lung, head & neck,ovarian, bladder, renal & testis, melanoma, lymphoma, liver, pancreaticand other gastrointestinal cancer. Specifically, desired cancer cellpopulation from various cancers include lung carcinoma cells, lungadenoma cells, colon adenocarcinoma cells, renal carcinoma cells, rectumadenocarcinoma cells, ileocecal adenocarcinoma cells, gastricadenocarcinoma, pancreatic carcinoma, hepatoma cells, hepatocellularcarcinoma cells, prostate adenocarcinoma cells, bladder carcinoma cells,breast carcinoma, ovarian teratocarcinoma, amalanotic melanoma cells,malignant melanoma cells, squamous cell carcinoma of the cervix, larynxand of oral origin; glioblastoma cells, endometrial adenocarcinoma,astrocytoma, Burkitt lymphoma cells, and non Hodgkin's lymphoma cells.

The presence of cancer cells bound to the matrix can be detected using avariety of different methods including the use of functional,immunophenotypic and cytomorphologic features of neoplastic cells.Additionally, any bound cell may be detected based on the ingestion ofthe labeled collagenous matrix by the cells.

5.2 Therapeutic and Preventive

Applications in the Present Invention

The present invention provides cell separation methods that may be used,for example, to remove undesired cancer cells and to enrichhematopoietic progenitor cells from the blood or bone marrow for use asdonor cells in bone marrow transplantation. Following cell separationmethods, antibodies reactive with carcinoma epitopes such as epithelialmarkers, or antibodies reactive with endothelial cells, such asanti-CD31 or anti-CD34, can be used to enrich for cancer or endothelialcell populations, respectively.

In a specific embodiment, cancer cells derived from different cancersmay be enriched from whole human blood by the cell separation methods ofthe present invention, subjected to ex vivo expansion, then used tointeract with dendritic cells to develop an effective tumor vaccineusing procedures including a method described (Brugger et al., 1999,Annals of the New York Academy of Science 872:363-371).

In another embodiment of the invention, circulating cancer cells can beisolated from a cancer subject and then subjected to a battery ofchemotherapeutic regimes in vitro to determine the efficacy of aspecific treatment. Effective doses or drug combinations could then beadministrated to that same subject (designer drugs).

In another embodiment of the invention, a cytotoxin-conjugated collagenmicrosphere can be injected into a patient with metastatic cancer forselective killing of cancer cells. Specific adhesion and internalizationof toxic collagen by cancer cells in vivo may provide a novel treatment.

The invention also provides fibrous compositions, including but notlimited to blood-borne adhesion molecule-coated collagen, fibrin, cottonand plastic fibers, to be used as cell-adhesion matrices for use asblood filters by subjects having metastatic cancer. The use of such acell-adhesion matrix involves the perfusion of the subject's bloodthrough the cell-adhesion matrix. In the blood perfusion protocol, thesubject's blood is withdrawn and passes into contact with thecell-adhesion matrix. During such passage, cancer cells present in thepatient's blood, preferentially adhere to the cell-adhesion matrix andare removed from the circulation of the patient.

To form the cell-adhesion matrix, collagen or the above-mentionedmaterial is formed within a vessel, including but not limited tocolumns, tissue culture plates, microcapillaries, or micromesh forcapture of cancer cells in biological fluids. The vessel contains aninput and output outlet for passage of the subject's blood through thecontainment vessel. In a specific embodiment of the invention, thecell-adhesion matrix may be formed within a containment vessel whichincludes a blood input line, which is operatively coupled to aconventional peristaltic pump. A blood output line is also included.Input and output lines are connected to appropriate arterial venousfistulas, which are implanted into, for example, the forearm of asubject. Alternatively, apheresed peripheral blood can be applied inconjunction with the above-mentioned cancer cell isolation by thecell-adhesion matrix. Apheresis was initiated upon recovery of the whiteblood cell count to equal or more than 1.times.10.sup.9/L. Apheresis orleukopheresis can be performed using a Cobe Spectra Cell Separator(Lakewood, Colo.) at a rate of 80 ml/min for 200 min (total volume of 16L). The use of the cell-adhesion matrix of the invention provides anovel method that can remove cancer cells from the circulation of apatient in which cancer cells have great potential to metastasize.

5.3 Assays for Measuring Metastatic Potential of Cancer Cells

It is an object of the present invention to provide a method for theidentification of subjects possessing a cancer with an increasedmetastatic potential. The present invention relates to evaluation ofmetastatic potential by detecting the ability of a subject's cancercells to invade, i.e., adhere, degrade and ingest a collagenous matrix,using the collagen based cell-adhesion matrix system of the presentinvention. The detection and measurement of the cancer cell's angiogenicpropensity, cell viability and proliferation constitute additional novelstrategies for prognosis of cancer.

The cell separation and assay methods described herein may be used for avariety of diagnostic uses. These uses include morphological, molecular,biochemical or immunological assays in early detection, in monitoringtherapeutic and surgical response, and in prognostication of cancerprogression. For example, RNA may be prepared from circulating cancercells and subjected to real-time polymerase chain reaction (PCR), DNAmicroarray analysis and serial analysis of gene expression (SAGE) and toidentify differentially expressed genes that are diagnostic markers forcancer or drug targets for controlling metastasis. Furthermore, proteinprofile of isolated cancer cells can be done by proteomics analyses todiscover novel metastatic markers and drug targets. The isolated cancercells may be stained for epithelial specific antigens, seprase and DPPIVantigens or endothelial cell markers, and quantitated to determine thenumber of cancer cells present for evaluating the metastatic potentialof a cancer. The isolated cancer cells may be assessed morphologicallyas a routine pathological evaluation of a cancer. The isolated cancercells may be cultured under sterile conditions and subjected tocytogenetic analysis to detect the presence of chromosomal abnormalitiesand mutation determination. The isolated cancer cells may be reactedwith molecular probes for more sensitive detection of mutation using DNAmicroarray, PCR and FISH. The methods thereby avoid the use of invasiveand expensive surgical procedures heretofore relied upon for such adetermination.

The present invention achieves a highly desirable objective, namelyproviding a method for the prognostic evaluation of subjects with cancerand the identification of subjects exhibiting a predisposition todeveloping metastatic cancer. Specifically, the invention encompassesfunctional assays for determining the metastatic potential of cancercells isolated from a subject using the cell-adhesion matrices of thepresent invention.

Specifically, the invention encompasses a method for determining themetastatic potential of cancer cells derived from a cancer subjectcomprising:

-   -   (a) inoculating a cancer cell sample derived from a cancer        subject onto a cell-adhesion matrix;    -   (b) incubation of the cancer cell sample for a time sufficient        to allow adhesion to, migration across or ingestion of the        matrix by the cancer cells to occur; and    -   (c) detection of adhesion to, migration across or ingestion of        the material by the cancer cells wherein detection of cancer        cell adhesion to, migration across or ingestion of the material        is an indicator of cancer cells with metastatic potential.

The assay of the invention involves the creation of an artificiallygenerated matrix onto which cancer cells are inoculated. Cells which maybe inoculated onto the matrix include but are not limited to bloodcells, cancer cell lines and cells derived from the tumor of a mammal,including cells derived from the tumor of a cancer subject, i.e.,biopsied tumor tissue, etc.

Once the cancer cells have been inoculated onto the matrix, the matrixis incubated for a time sufficient to allow adhesion, ingestion orinvasion to occur. The inoculation time will vary depending on themetastatic potential of the inoculated cancer cells.

Following incubation, cancer cell adhesion to the matrix and/oringestion of the matrix by cancer cells is detected using a variety ofdifferent methods. For example, a collagenous matrix may be labeled withagents such as, for example, fluorescent dyes, biotin, color dyes andradioactive probes. Ingestion of the matrix by the cancer cells resultsin labeling of the cells. The detection of cancer cells expressing thelabeled markers may be accomplished using a variety of different methodswell known to those skilled in the art including but not limited tofluorescence microscopy, fluorescent activated cell sorting (FACS) orscintillation counter measurement of radioactivity. Using such labels,the level of cell adhesion and ingestion of matrix material may bequantitated to determine the metastatic potential of the cells.

In addition, the metastatic potential of cancer cells isolated using thematrices of the invention may be determined using a variety of differentassays well known to those skilled in the art. For example, the cancercell sample can be incubated with labeled acetylated low densitylipoprotein (acLDL+), plated on a collagen gel for capillary networkformation, or stained with antibodies directed against endothelial cellmarkers, including but not limited to CD31+, Flk1+, and factor VIII(F8+) for a time sufficient to allow measurement of the acquirement ofthe endothelial phenotype by cancer cells to occur. Alternatively, thepropensity of the cancer cells to undergo apoptosis may be measuredusing an apoptosis assay kit (Molecular Probes, Inc.). The cancer cellclusters may be reacted with antibodies directed against leukocytemarkers, including but not limited to CD45, CD19, CD8, and CD4 for atime sufficient to allow numeration of cancer cell viability,association with immune cells (leukocytes, T-cells and killer cells) andresistance to cellular or complement cytolyses. The ability of isolatedcancer cells to form colonies in culture can also be determined bypropagating the cancer cells in tissue culture medium containing 10-20%human plasma for 1-5 weeks thereby allowing the cancer cells to formclones.

The assay system of the invention can also be used to monitor theefficacy of potential anti-cancer agents during treatment. For example,the metastatic potential of cancer cells can be determined before andduring treatment. The efficacy of the agent can be followed by comparingthe number or metastatic potential of the cancer cells throughout thetreatment. Agents exhibiting efficacy are those, which are capable ofdecreasing the level of detectable cancer cell adhesion, degradation oringestion of a collagenous matrix, the cancer cell viability, and thecolony-forming ability.

5.4 In Vitro Screening Assay for Identification of Anti-MetastaticAgents

The present invention further provides screening assays foridentification of agents capable of inhibiting the spread of cancercells from a primary tumor to a site of metastasis formation. Inaccordance with the invention, agents may be screened for their abilityto inhibit metastasis of cancer cells. In utilization of the assay ofthe invention for purposes of identifying anti-metastatic agents, thetest agent is co-inoculated with cancer cells onto the collagenousmatrix. The adhesion, ingestion and/or degradation of the collagenousmatrix by cancer cells in the presence of a test agent is compared tothe adhesion, ingestion and/or degradation of the collagenous matrix inthe presence of a vehicle control, wherein an anti-metastatic agent isidentified as one capable of inhibiting the adhesion, ingestion and/ordegradation of the collagenous matrix by cancer cells.

Specifically, the invention comprises a method for identifying an agentthat inhibits metastasis of cancer cells comprising:

-   -   (a) inoculating a cancer cell sample and either a test agent or        a vehicle control onto the cell-adhesion matrix    -   (b) incubation of the matrix for a time sufficient for adhesion,        degradation and/or ingestion of the matrix by the cancer cells        to occur; and    -   (c) detecting the adhesion and/or ingestion of the matrix by the        cancer cells,        wherein a decrease in the adhesion, ingestion and/or degradation        of the collagenous matrix by the cancer cells in the presence of        the test agent, as compared to the number of cancer cells        detected in the presence of a vehicle control, identifies a        compound that inhibits metastases formation.

The agents which may be screened in accordance with the inventioninclude, but are not limited to inorganic compounds, peptides,antibodies and fragments thereof, and other organic compounds (e.g.,peptidomimetics) that are capable of inhibiting the spread of cancercells from a primary tumor to a site of metastases formation. Agents mayinclude, but are not limited to, peptides such as, for example, solublepeptides, including but not limited to members of random peptidelibraries; (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84;Houghten, R. et al., 1991, Nature 354:84-86), and combinatorialchemistry-derived molecular library made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate directed phosphopeptide libraries; see,e.g., Songyang, Z. et. al., 1993, Cell 72:767-778).

Agents identified via assays such as those described herein may beuseful, for example, in defining the properties of cancer cells thatenable successful migration and invasion, and for inhibiting metastasesformation in cancer subjects. Assays for testing the efficacy ofcompounds identified in the screens can be tested in animal modelsystems for metastasis formation. Such animal models may be used as testsubstrates for the identification of drugs, pharmaceuticals, therapiesand interventions which may be effective in treating cancer metastasis.

The assay of the invention may also be used to screen for nucleic acidsequences capable of inhibiting the metastatic potential of cancercells. Such nucleic acid molecules include molecules comprising proteincoding sequences or anti-sense sequences. The nucleic acid molecules maybe transferred to cancer cells prior to assaying by such methods aselectroporation, lipofection, calcium phosphate mediated transfection,or viral infection.

In accordance with the invention, an assay system can be used to screenfor agents that modulate the activity of serine integral membraneproteases, including DPPIV and seprase and thereby modulate themetastatic potential of cancer cells. To this end, cells thatendogenously express DPPIV and seprase can be used to screen for agents.Alternatively, cell lines, such as 293 cells, COS cells, CHO cells,fibroblasts and the like are genetically engineered to express DDPIV andseprase for use in screening. Preferably, host cells geneticallyengineered to express DDPIV and seprase can be used as an endpoint inthe assay; e.g., as measured by a chemical, physiological, biological orphenotypic change.

In utilizing such cell systems, the cells expressing the DDPIV andseprase are exposed to a test compound or to a vehicle control. Afterexposure, the cells can be assayed to measure the expression and/oractivity of DDPIV and seprase protease activity. For example, afterexposure, cell lysates can be assayed for gelatinolytic activity(seprase), or substrates containing phosphorylated residues adjacent toproline, such as H-Gly-Pro-p-nitroanilide or amino methylcoumarin (DPPIVand seprase) and Z-Gly-Pro-p-nitroanilide or amino methylcoumarin(seprase) (Kaspari et al., 1996). The ability of a test compound todecrease levels of DPPIV and/or seprase protease activity, below thoselevels seen with cells treated with a vehicle control, indicates thatthe test compound inhibits DDPIV and seprase associated proteaseactivity.

In addition, assays may be used to identify agents that antagonize theinteraction between DPPIV and seprase, thereby inhibiting the activityof the protein complex. To identify such agents, DPPIV and sepraseproteins are incubated in the presence and absence of a test compoundfollowed by detection of complex formation. A decrease in complexformation indicates the identification of an agent capable ofantagonizing the interaction between DPPIV and seprase.

In one aspect of the invention, soluble DPPIV and seprase may berecombinantly expressed, labeled and utilized in none-cell based assaysto identify compounds that inhibit the interaction between DPPIV andseprase. In such assays, either the DPPIV or seprase can be attached toa solid substrate such as a test tube or microtitre well, by means wellknown to those known in the art. The test agents are then assayed fortheir ability to inhibit the interaction between DPPIV or seprase on thesolid substrate.

5.5 Compositions and Uses

The present invention relates to a novel cancer cell capture system forrapid and efficient detection and selection of invasive cancer cellsfrom the blood and/or tissue of cancer patients. The invention relatesto fibrous compositions comprising blood-borne adhesion molecule-coatedcollagen, fibrin, cotton, and plastic fibers, to be used ascell-adhesion matrices. The invention further relates to use of suchcoated fibers to detect the presence of metastatic cells in cancerpatients, and to filter the blood of subjects having metastatic cancers.Cell-adhesion matrices that may be administered to subjects with cancerinclude those described in Sections 5.1.-5.2., supra.

The present invention also provides cellular compositions that can beisolated by the cell-adhesion matrix of this invention. Such cellularcompositions comprise a small subpopulation of tumor cells that havepotential to metastasize, as well as cancer cell clusters in the bloodthat may contribute to organ dysfunction associated with late stagecancer. The enriched cancer cell population can be used, for example, todetermine novel drug targets, genetic defects involved, their metastaticpotential and the most effective treatment regime. The enriched cellsmay also be used in fusions with dendritic cells for cancer vaccinedevelopment.

The present invention provides for treatment of proliferative disorderssuch as cancer, by administration of agents that regulate the metastaticactivity of cancer cells. Compounds that may be administered to subjectswith cancer include those agents identified using the assays describedin Sections 5.3., supra.

In a specific embodiment of the invention, key proteases required forthe tissue invasive phenotype were identified. For example, activationof serine integral membrane proteases, such as seprase and DPPIV wereshown to be required for tissue cell migration and invasion. Thusinhibitors of such proteases may be used to inhibit the metastaticactivity of cancer cells. Such inhibitors include antibodiesimmunospecific for seprase or DPPIV (FIGS. 13A-E). Alternatively,transfection into cancer cells of a vector expressing antisense RNA orribozymes for seprase or DPPIV may be used to reduce the ability ofcancer cells to metastasize (FIG. 19).

In addition, protease inhibitors may be administered to inhibit thespread of cancer cells from a primary tumor to a site of metastasisformation. As demonstrated in Section 7, Example 2, an inhibitor ofDPPIV, i.e., an anti-DPPIV antibody, was capable of inhibiting cancercell migration. Cancers involving metastasis of tumor cells to otherlocations in the body are treated by administration of an agent thatinhibits cancer cell migration and tissue invasion. Such cancersinclude, for example, carcinomas, such as breast cancer and prostatecancer.

Compounds identified for use in prevention of cancer cell metastasisescan be tested in suitable animal model systems prior to testing inhumans, including but not limited to dogs, rats, mice, chicken, cows,monkeys, rabbits, etc. For in vivo testing, prior to administration tohumans, any animal model system known in the art may be used.

In specific embodiments, compounds that inhibit cancer metastasis areadministered to a subject having cancer where it has been determinedthat the subject's cancer cells have an increased metastatic potential.The increased metastatic potential can be readily detected, e.g., byobtaining blood sample (or biopsy tissue from a patient) and assayingfor the metastatic potential of the isolated cancer cells using the cellseparation and assay system of the present invention.

The invention provides methods of treatment of cancer by administrationto a subject of an effective amount of a compound that inhibits themetastatic potential of cells. In a preferred aspect, the subject is ananimal, and is preferably a mammal, and most preferably human.

The present invention also provides pharmaceutical compositions that canbe linked to the cell-adhesion matrix or its isolated cellular componentof this invention. Such compositions comprise a therapeuticallyeffective amount of the compound capable of regulating the migration andinvasion of cancer cells, and a pharmaceutically acceptable carrier. Ina specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the compound is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.

6. EXAMPLE Isolation of Circulating Carcinoma Cells Involved inAngiogenesis and Metastasis

The data provided below, demonstrates that viable carcinoma cells can beisolated from peripheral blood of cancer patients with metastaticdiseases by novel cell-adhesion matrix cell separation and assaymethods, and these cells acquire molecular determinants necessary forangiogenesis and metastasis to occur. Comparing to the cells derivedfrom primary tumor, such viable circulating cancer cells represent asmall subset of cancer cell population that have potential tometastasize.

6.1 Materials and Methods 6.1.1 Collection of Blood Sample

Blood or cells collected from an in vivo source are subjected to cellisolation within a relatively short time after their collection becausethe cells may lose their viability. In order to maintain the optimalisolation of cancer cells, it is preferred that blood, ascites or tissuesamples are stored at 4° C. and used within 24 hours after theircollection, most preferably, within four hours. Approximately 10-20 mlof blood each time were collected in Vacutainer tubes (Becton Dickinson,green top, each tube holds 7-ml) containing Lithium Heparin asanticoagulant. Patient age, sex, date of diagnosis, therapeuticinterventions, clinical status, and biopsy report were retrieved fromthe patients' charts. The protocol was approved by the institutionalreview boards.

6.1.2 Isolation of Cancer Cells from Blood or Tumor Tissue

The collagen polymerization solution was prepared and adjusted to apre-determined concentration, from 1 to 2 milligrams per milliliter forcollagens in Dulbecco's modified Eagle's medium (DMEM), on iceimmediately prior to the gelation into vessel substrata (FIG. 1A).Specifically, type I collagen solution (rat-tail type I collagen, 4.0mg/ml, Collaborative Biomedical Products, Becton and Dickinson Labware,Bedford, Mass.) was mixed with equal volume of DMEM at 4° C. The mixturewas overlaid as a thin layer onto the bottom of 96-well microtiter or6-well culture plates (Nunc, Inc., Naperville, Ill.) at 4° C. To form agel of collagen fibers, plates were incubated for 30 minutes at 37° C.to allow polymerization of collagen layer.

6.1.3 Isolation of Cancer Cells from Blood, Ascites or Tumor Tissue

To separate cancer cells from a cell population, the collagen matrix wasfirst coated with the cell culture medium, consisting of a 1:1 mixtureof Dulbecco's modified Eagle's medium (DMEM) and RPMI1640 supplementedwith 10% calf serum, 15% Nu-serum (Collaborative Research, Inc.,Bedford, Mass.), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1mM sodium pyruvate, 1 unit/nil penicillin, and 10 μg/ml streptomycin.However, cancer cells can be isolated directly from whole blood withoutthe need to coat additional adhesion molecules, which are present in theblood.

The samples were processed for plasma and cell isolation at 4° C. Bloodsamples were centrifuged to collect plasma, and cell pellets werediluted with PBS containing 2% bovine serum and 0.5 mM EDTA to theoriginal volume and then fractionated using Ficoll-Paque (Pharmacia) tocollect mononuclear cells. The mononuclear cell fraction was furtherselected for viable and invasive carcinoma cells by differentialadhesion to the cell-adhesion matrix. Briefly, mononuclear cells weresuspended in the complete culture medium (to same volume as the blood).A portion of mononuclear cells were seeded, i.e., 0.1-ml per well of96-well microtiter plate or 1 to 10 ml per well of 6-well tissue cultureplate (NUNC) that were coated with the collagen matrix, for 15 minutesto 1 hour. The culture was washed gently with medium to removenon-adherent cells. For immunocytochemistry and functional assays, cellsin microtiter plates were cultured for 12-24 hours before processing.For cell separation studies, cells adherent on the matrix were thensuspended with trypsin/EDTA solution (GIBCO) for 5 minutes or simply byvigorously washing with phosphate buffered saline (PBS). Cells in thewashes were transferred into a 6-well tissue culture plate and culturedfor 12 hours to 24 days in a CO₂ incubator at 37° C. It should be notedthat, in case of patients with high white blood cell count, i.e.,patients with lymphoma or leukemia, the mononuclear cell fraction shouldbe diluted with the complete medium to 1-2 million cells per ml for onewell of 6-well tissue culture plate. Recovery of cancer cells isdependent on cell density on the matrix (see below). Viability of thecells was evaluated by Trypan Blue exclusion or apoptosis assays.

Alternatively, whole blood is deprived directly of viable and invasivecarcinoma cells by passing through cell culture beads coated with acell-adhesion matrix such as type I collagen or filters made of purifiedcotton. A sterile 30-ml pipette was packed with purified cotton orcollagen-coated beads, and 0.1-ml of collagen-coated bead was used forevery 10-ml blood that was pre-diluted with equal volume of mediumcontaining 0.5 mM EDTA or with 10% anticoagulant citrate dextrose (ACD,Bakter Healthcare Corporation, Ill.) containing 50 units heparin/mL. Thecolumn was pre-washed by medium containing 10% human plasma or serum.Flow rate, from 0.1 to 0.7 ml per minute, results in capture of cellsthat share similar features as these derived from the substratum methoddescribed above.

6.1.4 Labeling of Collagen Matrix and Measurement of the Cell InvasivePhenotype

Collagen was polymerized prior to biotin, fluorescein or rhodaminelabeling so that sites of polymerization were not perturbed. Labeledcollagen fibers were then solubilized in acidified water (pH 2.0), butcould be readily polymerized back to collagen fibers under experimentalconditions. Specifically, 10 ml type I collagen solution (rat-tail typeI collagen, 4 mg/ml, Collaborative Biomedical Products, Becton andDickinson Labware, Bedford, Mass.) was mixed with 10-ml DMEM and addedinto a 10 centimeter tissue culture plate at 4° C. The plate wasincubated for 30 min at 37° C. to allow polymerization of the collagenfibers (gel). The gel was washed with 30 ml of coupling borate buffer,pH 9.3 (Sigma) for 30 min and then incubated with 30 ml borate buffercontaining 3 mg of Sulfo-NSH-Biotin (Pierce), Fluorescein IsothiocyanateI hydrochloride (FITC), Tetramethyl Rhodamine Isothiocyanate (TRITC)(Research Organics Inc, Cleveland, Ohio) or 1 mg of Bodipy-rhodamine orfluorescein dyes (Molecular Probes, Inc.), at 25° C. on a shaker.Conjugation was stopped by washing 3 times with PBS, followed by a 50-mlPBS washing for 2 days and a 50-ml distilled water wash for another 2days. Labeled collagen fibers were solubilized in acidic water (0.02Nacetic acid) to a final concentration of 1 mg/ml. Labeled collagenmonomers were mixed with equal volume of unlabeled collagen solution,and further diluted with 2× volume of DMEM, overlaid on vessel surfacesto form a thin layer, and incubated for 30 min at 37° C. to allow gelformation.

Labeled collagenous matrix was coated on a 16-well microtiterplate-glass slide (in 96-well microtiter plate format; Lab-Tek,Rochester, N.Y.) or 6-well tissue culture plate (NUNC). A portion ofmononuclear cells, i.e., 0.1-ml per well of 96-well microtiter plate or1.6-ml per well of 6-well tissue culture plate (NUNC), were seeded onsuch substratum and cultured for 15 minutes to 1 hour to captureadherent cells. After washing of non-adherent cells, the cells weregrown on the labeled matrix for 12 to 24 hours for measurement ofcellular invasiveness using Nikon Eclipse E300 inverted light microscopein conjunction with SONY DC5000 Cat Eye Imaging system or MolecularDevices fMax Fluorescence Microplate Reader in conjunction withSOFTmaxPRO 1.2F for Windows software computer analysis as depicted inFIGS. 1A, 1B. Initially, the Bodipy-fluorescent collagen substratadescribed above were prepared in 16-well culture chambers to optimizecell culture and microscopic imaging analysis conditions forcharacterization of the invasiveness of the matrix-isolated cells (FIG.1A). Substrate preparations were then optimized for 96-well microtiterplate version. Optimization parameters include feasibility for samplethroughputs, and resolution for accuracy of quantitation.

6.1.5 Other Cellular Function Assays

A functional assay was performed for endothelial cell activity usingacetylated low-density lipoprotein (acLDL) Bodipy FL conjugate asdescribed in manufacture manual (L-3485, Molecular Probes, Oreg., USA).To resolve blood cells co-isolated on the matrix that were apoptotic ornecrotic, the cells were stained prior to fixation using VybrantApoptosis Assay Kit #5 Hoechst/prodidium iodide (V-13244, MolecularProbes, Oreg., USA).

6.1.6 Cell Lines and Culture

The human breast carcinoma cell lines MDA-MB-436 and Hs578T wereobtained from American Type Culture Collection (Rockville, Md.), and thehuman amelanotic melanoma cell line LOX was obtained from ProfessorOystein Fodstad, Institute for Cancer Research, The Norwegian RadiumHospital, Oslo, Norway. Cells were cultured in a 1:1 mixture ofDulbecco's modified Eagle's medium (DMEM) and RPMI1640 supplemented with10% calf serum, 5% Nu-serum (Collaborative Research, Inc., Bedford,Mass.), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodiumpyruvate, 1 unit/ml penicillin, and 10 μg/ml streptomycin. These cellswere used to evaluate the reagents for immunocytochemical detection andto determine sensitivity of functional assays.

6.1.7 Sample Preparation for Immunocytochemistry

In order to enumerate transformed epithelial cells in the blood ascompared to leukocytes and peripheral blood tissue cells, themononuclear cells derived from blood of cancer patients that werecaptured by labeled collagenous matrix coated on a 16-well chamberedglass slides (Lab-Tek, Rochester, N.Y.) and cultured on the samesubstratum for 12-24 hours in a CO₂ incubator at 37° C. were fixed forimmunocytochemistry. Primary mAbs used in this study include mouse mAbrecognizing human epithelial specific antigen (ESA; clone VU-1D9,NeoMarkers, Calif., USA; SIGMA, MS, USA), Muc-1 epithelial membraneglycoprotein (Muc-1; clone E29, NeoMarkers, Calif., USA), cytokeratins4,5,6,8,10,13, and 18 (PCK; clone C-11, SIGMA, MS, USA); CD31/PECAM-1endothelial cell marker (CD31; Clone JC/70A, NeoMarkers, Calif., USA),Flk-1, a receptor for vascular endothelial growth factor (Flk-1, Clonesc-6251, Santa Cruz, USA), VE-cadherin endothelial marker (VE-cad; Clonesc 9989, Santa Cruz, USA); CD34 peripheral blood stem cell marker (CD34;clone 581, Pharmingen, USA), CD45 leukocyte common antigen (CD45; cloneT29/33, DAKO, Denmark), CD8 suppressor T cell marker (CD8; clonec8/144B, NeoMarkers, Calif., USA), CD43 T cell marker (CD43; clone84-3C1, NeoMarkers, Calif., USA), prostate specific acid phosphatase(PSAP; clone PASE/4LJ, NeoMarkers, Calif., USA), prostate specificantigen (PSA; clone ER-PR8, NeoMarkers, Calif., USA), c-erbB-2/Her-2/neuoncoprotein (erB-2; clone e2-4001+3B5, NeoMarkers, Calif., USA),c-erbB-2 (Clone TAB250, Zymed, Calif., USA), CA 19-9/sialyl Lewis GItumor marker (CA19-9; clone 121 SLE, NeoMarkers, Calif., USA), or p53tumor suppressor protein (p53; clone DO-7+BP53-12, NeoMarkers, Calif.,USA). In addition, fluorescein conjugated antibodies against Muc-1epithelial cell marker (DAKO, Denmark) and fluorescein conjugates ofgoat antibodies against factor VIII endothelial marker (F8; Atlantic)were used to doubly stain carcinoma and endothelial cells, respectively,in addition to above primary antibodies against other cell markers.Furthermore, rat mAbs D28 (against seprase), E19, E26 (against DPPIV)and C27 (against β1 integrin) generated in our laboratory were used.

Antibody staining involves the addition of primary antibody and/orfluorescein-F8 or -Muc-1 to the slides after blocking nonspecificbinding sites with 2% BSA for 30 min. The samples were incubated for 20min at room temperature, washed twice in PBS for 5 min, and then exposedto secondary rabbit anti-mouse Ig (Z0259, Dako) for another 20 min.After two more washes, the samples were incubated withalkaline-phosphatase-anti-alkaline-phosphatase (APAAP) mouse Igcomplexes for 15 min. Finally, the enzyme-substrate [NewFuchsin (Dako)]was added, resulting in the development of red precipitates. The datawere recorded by using Nikon Eclipse E300 inverted light microscope inconjunction with SONY DC5000 Cat Eye Imaging system and were stored on acomputer server for later reference.

6.1.8 Sample Preparation for Flow Cytometric Analysis

In order to enumerate transformed epithelial cells in the blood thatwere captured by labeled collagenous matrix coated on a 6-well tissueplate, the mononuclear cells released from the matrix substratum wereanalyzed by flow cytometry following a manufacture's procedure. Similarto procedures involved in immunocytochemistry, the cells were determinedfor apoptosis or necrosis by staining prior to fixation using VybrantApoptosis Assay Kit #5 Hoechst/prodidium iodide (V-13244, MolecularProbes, Oreg., USA). Briefly, the mononuclear cells were stained in asolution containing fluorescein (FITC)-conjugated mouse mAb C11 againstcytokeratins 4, 5, 6, 8, 10, 13, 18 (PCK; Sigma) orfluorescein-antibodies against Muc-1 (DAKO), phycoerythrin(PE)-conjugated anti-CD31 endothelial marker (CD31; Becton-Dickinson)and peridinin chlorophyll protein (PerCP)-labeled anti-CD45 (CD31;Becton-Dickinson) for 15 min. After incubation and washing, thecollected cells were resuspended in 0.5 ml of a buffer and the samplewas analyzed on a FACScan or FACSVantage flow cytometer (BectonDickinson).

6.2 Results 6.2.1 Isolation of Cancer Cells Using theCell-Adhesionmatrix: Role of Blood Borne Cell Adhesion Molecules

Table I shows the attachment of LOX human malignant melanoma cells andcancer cells isolated from a patient with head-neck cancer to variousnatural fiber surfaces in the presence of human plasma fibronectin,laminin and vitronectin, as well as human and bovine sera. Attachment ofhuman cancer cells were assayed in 96-well plates (Nunc, Inc.,Naperville, Ill.) coated with either various amounts of purified serumproteins (0.1 to 1 μg per well) or 10% serum and was performed asdescribed (Nomizu et al., 1995). Cell attachment properties of naturalfibers were determined by counting the number of cells in the area thatremained attached on 1.27 mm² areas of a microtiter well. In thepresence of human or bovine sera or fibronectin+laminin+vitronectin, LOXmelanoma cells adhere better on purified collagen, fibrin, and cottonfibers, as well as lined plastic surfaces than plain plastic, and havehigher numbers of cells that remain attached (Table 1). Similarly,head-neck cancer cells attach better on purified collagen, fibrin, andcotton fibers, as well as lined plastic surfaces than plain plastic, andhave higher numbers of cells that remain attached (Table 2). Theseresults suggest that the surfaces of the cell-adhesion matrix exerttheir effects on cancer cell adhesion through their binding affinity forblood-borne adhesion molecules.

TABLE 1 Attachment of LOX melanoma cells on various cell-adhesion matrixsurfaces is mediated primarily by binding of the matrix to blood-bornecell adhesion molecules. Human Bovine serum serum FN + LM + VN Negativecontrol Type I collagen 92 ± 5 91 ± 6 86 ± 5 8 ± 5 Fibrin 68 ± 7 72 ± 867 ± 6 6 ± 6 Purified cotton 79 ± 5 83 ± 9 72 ± 6 7 ± 6 Lined plastic 57± 8 49 ± 9 35 ± 8 4 ± 3 Plain plastic  7 ± 3  5 ± 4  3 ± 2 2 ± 1

Cells, 4×10³ per well (the number of cells in the area that remainedattached on 1.27 mm² areas of a microtiter well was counted), wereseeded on various matrix surfaces in 96-well plates (Nunc, Inc.,Naperville, Ill.), that were coated with 10% human serum (Sigma), 10%bovine serum (Collaborative Research, Inc., Bedford, Mass.), or humanplasma fibronectin+laminin+vitronectin (FN+LM+VN; Gibco-BRL,Gaithersburg, Md.) at the concentration of 10 μg/ml each. The wells thatwere not coated with protein were used as negative control. Type Icollagen fibers were formed on the bottom of microtiter wells accordingto the procedure described below. Fibrin fibers were made by clotting20% human plasma in Dulbecco's modified Eagle's medium in microtiterwells. Purified cotton fibers were also suspended in Dulbecco's modifiedEagle's medium and seeded in microtiter wells. “Lined plastic” indicatesthat the surface of microtiter wells had been scrapped with an Eppendorfpeptide tip. Each value represents the mean±S.D. of three independentexperiments.

TABLE 2 Attachment of head-neck cancer cells on various cell-adhesionmatrix surfaces is mediated primarily by binding of the matrix toblood-borne cell adhesion molecules. Human Bovine serum serum FN + LM +VN Negative control Type I collagen 42 ± 4 41 ± 4 37 ± 5 2 ± 2 Fibrin 37± 3 33 ± 5 27 ± 3 3 ± 2 Purified cotton 38 ± 5 34 ± 4 29 ± 2 2 ± 1 Linedplastic 27 ± 2 28 ± 3 22 ± 2 2 ± 1 Plain plastic  5 ± 2  3 ± 2  2 ± 1 2± 1Cells, 2×10³ per well (the number of cells in the area that remainedattached on 1.27 mm² areas of a microtiter well was counted), wereseeded on various matrix surfaces in 96-well plates (Nunc, Inc.,Naperville, Ill.), that were coated with 10% human serum (Sigma), 10%bovine serum (Collaborative Research, Inc., Bedford, Mass.), or humanplasma fibronectin+laminin+vitronectin (FN+LM+VN; Gibco-BRL,Gaithersburg, Md.) at the concentration of 10 μg/ml each. The wells thatwere not coated with protein were used as negative control. Type Icollagen fibers were formed on the bottom of microtiter wells accordingto the procedure described below. Fibrin fibers were made by clotting20% human plasma in Dulbecco's modified Eagle's medium in microtiterwells. Purified cotton fibers were also suspended in Dulbecco's modifiedEagle's medium and seeded in microtiter wells. “Lined plastic” indicatesthat the surface of microtiter wells had been scrapped with an Eppendorfpeptide tip. Each value represents the mean±S.D. of three independentexperiments.

6.2.2 Sensitivity of Cell Separation Methods

The human invasive breast carcinoma cell lines MDA-MB-436 and Hs578T(American Type Culture Collection, Rockville, Md.) were used todetermine sensitivity of the cell separation method and to evaluate thereagents for immunocytochemical detection (see below). These breastcancer cells were tagged with a fluorescent dye to determine thesensitivity of the isolation procedure. FIGS. 3A-G show the analysis ofnormal and bladder cancer blood samples to which varying numbers ofHs578T breast carcinoma cells, which were tagged with PKH26 RedFluorescent Cell Linker (Sigma), were added. As can be seen, thecarcinoma cells can be distinguished from the other blood cells. Therecovery of breast carcinoma cells was consistent over a frequencyrange, between 1 and 500 Hs578T cells spiked into 1-ml of blood (10-20million mononuclear cells per ml) from normal donors, and the recoveryrate was between 75 and 100%. However, when Hs578T breast carcinomacells were spiked into the blood from a bladder cancer donor with lessthan 10 million mononuclear cells per ml, the recovery of the Hs578Tcells was between 95 and 100%, suggesting cell density is a limitingfactor for cell adhesion to the matrix. In addition, when 19 redfluorescent tagged Hs578T cells were spiked into 1-ml of blood from abladder cancer donor, both isolated Hs578T and bladder cancer cells wereidentified by positive staining with anti-cytokeratin antibody C11(FIGS. 3E-3F). There were 16 fluorescent tagged Hs578T cells and 468bladder carcinoma cells detected in one ml blood. These results suggestthat the level of sensitivity by the matrix capture method is at 1viable cancer cell per ml of blood, and the recovery rate can reach 85%.

6.2.3 Cancer Cell Isolation Using the Cell-Adhesion Matrix

For detection of cancer cells in blood, a thin layer ofrhodamine-collagen solution coated on 16-well chambered glass slides(Lab-Tek, Rochester, N.Y.) or 96-well culture plates (NUNC), which issubsequently incubated with calf serum containing blood-borne adhesionmolecules, is a preferred embodiment of the “matrix”. Mononuclear cellsin buffy coat derived from 1.6 millimeter (ml) of whole blood are seededin equal aliquots, 0.1 ml each, into each well of a 16-well chamberedglass slide, and incubated for 10 minutes to 18 hours at 37° C. toselect for cancer cells by differential adhesion to the collagen matrix.

FIGS. 4A-D depict the isolation of circulating cancer clusters withtissue fragments using such a type I collagen based, cell-adhesionmatrix. Such cell clusters of various sizes were commonly observed inthe blood of patients with advanced metastatic disease, includingsquamous cell carcinoma of head and neck (FIGS. 4A-C) and prostatecancer (FIG. 4D). It has been estimated that there are large numbers ofcancer cell clusters in the blood of stage III-IV cancer patients,ranging from a few hundred to twenty thousand per cubic centimeter ofblood. However, there are no such cell clusters found in the blood ofnormal donors or early stage cancer patients. Furthermore, fibrin fibers(FIG. 4E), plastic scraps (FIGS. 4F-G), purified cotton fibers (FIGS.4H-I), and type I collagen fibers (FIGS. 5A-H) capture circulatingcarcinoma cells that grow in culture and exhibit epithelial cell shape.Surfaces of these materials absorb blood-borne cell adhesion molecules,which in turn promote the attachment and spreading of tumor cells (seeTable II above). The role of cell adhesion molecules in this process hasbeen further demonstrated by incubation of coated collagen matrix withpolyclonal antibodies directed against fibronectin, vitronectin, andlaminin. This treatment resulted in a reduction of cell capturecapability of the matrix.

FIGS. 5A-H show the morphology of circulating cells and their underlyingtype I collagen-based, cell-adhesion matrix in the presence ofblood-borne cell adhesion molecules. These carcinoma cells adherepreferentially to the collagen matrix within 10 minutes to 60 minutesafter contact. They then degrade and ingest the matrix generating holeson flat substrata, followed by migration and growth on the plasticsurface (FIGS. 5A-D; 6A-D). Such carcinoma cells are not present inblood of most normal donors (FIG. 7A), patients with benign disease(FIG. 7B), or cancer patients undergoing chemotherapy (FIGS. 7C-D).Freshly isolated circulating cancer cells are small in size butrelatively larger than most of mononuclear blood cells co-isolated onthe matrix (FIG. 6A). These putative cancer cells grow rapidly, becomelarger in size, and assume epithelial shape within four days in culture(FIGS. 5A-D; 6A-D). However, co-isolated leukocytes are small and reducetheir number in culture (FIGS. 5A-D). Also, few carcinoma cells isolatedfrom squamous cell carcinoma of head and neck biopsies (SCCHN) using thecell-adhesion matrix (FIGS. 5E-F) exhibited the invasive phenotype orgrowth characteristics of circulating cancer cells, suggesting thatcirculating cancer cells represent a unique subset of carcinoma cellpopulation that metastasizes. In such SCCHN tissue, they are fibroblaststhat invade the collagen gel and propagate in culture (FIGS. 5E-F). Itshould be noted that the cell separation system captures cells whichremain viable, i.e., cells which retain the ability to adhere, but notthose that are damaged in the circulation or by experimentalmanipulation.

The thickness of the collagen matrix was estimated approximately 100 μm,as determined by both light microscopic and transmission electronmicroscopic measurements (FIGS. 5E-H). The assembled collagen cancapture cancer cells from mixed cell population, i.e., blood or cellsuspension derived from tissue biopsy, within 10 minutes to one hour.The matrix-captured cells can further invade into the matrix during theone to two week incubation, leaving behind some non-invasive cells,which do not multiple, on the surface of the matrix (FIGS. 5E-F). Theassembled collagen fibers are fine strings of 70 nm in diameter and havesimilar appearance as these in the collagen bundles in vivo except theylack the collagen-banding pattern (FIGS. 5G-H).

6.2.4 Functional and Immunophenotypic Features of Circulating CancerCells

The functional and immunophenotypic features of the epithelial cellscaptured by the matrix are consistent with those of neoplastic cells.For measurement of the invasive phenotype of these cells, cells thatwere isolated from blood of cancer patients using rhodamine-collagenmatrix were analyzed for the ability of the cell to adhere to, degradeand ingest rhodamine-collagen substratum. FIGS. 8A-I show that putativecarcinoma cells exhibit extensive collagen-degrading/ingestionactivities (Col+); these cells also exhibit immuno- and morphologicalfeatures characteristic of carcinoma cells (see below).

For the determination of nature of cancer cells, cells that wereisolated from blood of cancer patients using rhodamine-collagen matrixwere analyzed for their potential epithelial origin byimmunocytochemistry using antibodies against epithelial specific antigen(ESA), epithelial membrane antigen (Muc-1), and cytokeratins 4, 5, 6, 8,10, 13, and 18 (PCK). These cells react positively with ESA+, Muc-1+, orPCK+ are epithelial origin, and therefore, consistent with theirmetastatic nature. On the matrix, adherent cells including enrichedcancer cells are stained with cell type-specific monoclonal antibodiessuch as these against epithelial, carcinoma, endothelial, peripheralblood stem cells, or leukocytes. Adherent cells, that are labeledpositively for epithelial cell markers (FIGS. 8E, G, J, K) and thatingested collagen fragments, Col+ (FIGS. 8B, C, F, I, L), are identifiedas carcinoma cells. Circulating carcinoma cells are rare in blood ofmost normal donors (FIG. 8L), patients with benign disease, or cancerpatients undergoing chemotherapy. Similar to immunocytochemical analysispresented here, mononuclear cells derived from blood of cancer patientsthat were released from the matrix can be subject tofluorescence-activated cell sorting (FACS) analysis.

6.2.5 The Acquisition of an Endothelial Cell Phenotype by CirculatingCarcinoma Cells

In addition to invasion, metastatic carcinoma cells in the circulationinvolve an apparently novel angiogenic propensity. Circulating carcinomacells isolated from the blood of cancer patients using a cell-adhesionmatrix inevitably acquire endothelial characteristics. These squamouscell carcinoma of head-neck (HN, FIGS. 9A-C; 9I-J), colon cancer (CC,FIGS. 9D-E) and prostate cancer (PC, FIGS. 9G-H) cells are Col+ (FIGS.9C, F), stain for the endothelial cell markers, including von Willebrandfactor or factor VIII (F8) (FIGS. 9B, E) and CD31 (FIG. 9D), and expressan endothelial cell ability by incorporating acetylated low-densitylipoprotein (LDL) (FIGS. 9H, J) as well as an epithelial surface antigen(ESA in FIG. 9G). In addition, when the isolated carcinoma cellsinternalized and were labeled with fluorescein-LDL and then plated on acollagen gel of 0.5-mm in thickness, they showed enhanced endothelialdifferentiation, including the formation of cellular networks andtube-like structures (FIGS. 9K, L). These structures consistedprincipally of LDL-labeled carcinoma cells. Furthermore, circulatingcarcinoma cells illustrate additional endothelial features including theexpression of the receptor for vascular endothelial growth factor Flk-1and VE-cadherin, and without loss of previously expressed epithelialmarkers. This angiogenic propensity is restricted to those carcinomacells that enter the circulation and not with cells that remain in thetumor tissue. Such spatial restriction in angiogenic phenotype reflectsdistinct functional abilities of circulating carcinoma cells toextravasate, colonize and cooperate with angiogenesis that lead toformation of micrometastases.

6.2.6 Immune Cancer Killing and Growth of Circulating Cancer Cells inCulture

Circulating cancer cells form clusters with white blood cells (WBC) thatexpress the leukocyte common antigens CD45 and the T-cell marker CD8.The cell-adhesion matrix could readily isolate such clusters of immuneand cancer cell complex from cancer patients who exhibited encouragingprognosis. Although total number of circulating cancer cells mayindicates a potential for metastasises formation, numerations of thecancer cells that interact with cytotoxic WBC (FIGS. 10A-I) orautoimmune anti-cancer antibody-mediated, complement cytolysis (FIGS.10J-L) signify the host immunity against metastasis. In these cancerpatients who exhibited favorable prognosis, most cancer cells wereattacked by leukocytes and became fragmented after one day in cultureand only a few cancer colonies grew (FIGS. 10F-I). In addition,anti-cancer antibody-mediated complement cytolysis can take place inmost cancer patients. Such killing of cancer cells was demonstrated hereby culturing isolated BLC cells in the presence of 10% autologousplasma, derived from the blood of the same cancer patient (FIGS. 10J-K),but not in those from a normal donor (FIG. 10L). Resulting cytolysis ofcancer cells can be determined by morphological appearance of phase-darkmaterials in lysed cells (FIGS. 10J-L). Importantly, approximately 97%cells freshly isolated from the blood are apoptotic as determined byfluorescent staining using apoptosis and cell lyses kits produced byMolecular Probes, Inc. Although some cancer cells can propagate in theculture for two months, most cancer cells show apoptosis or cytolysisimmediately after their isolation, indicating cancer cell killing byhost immunity. Most WBC disappear from the culture after one week,leaving behind clusters or colonies of cancer cells (FIGS. 10F-L). Thus,numeration of viable and invasive cancer cells that resist immunekilling would be the strongest indicator for patients who process highdegree of malignancy.

6.2.7 Enumeration of Circulating Cancer Cells

Circulating cancer cells isolated using the cell separation methods wereanalyzed for their invasive activities, epithelial nature, angiogenicpropensity, and resistance to host immunity as described above. Resultswere recorded using an inverted Nikon microscope computerized with aSONY DKC5000 3CCD imaging system. Numeration of cancer cells present inwhole blood or buffy coat by this cell separation and assay methodsdescribed herein presents the highest sensitivity and resolution. Over200 blood samples of patients with prostate, breast, colon, lung, head &neck, brain, bladder, lymphoma, renal & testis, liver, or pancreatic andother gastrointestinal cancers were examined. Circulating cancer cells,as defined by Col+, LDL+, ESA+, Muc-1+, PCK+, F8+, or CD31+ cells, arein the order of two thousand to twenty thousand per ml of whole bloodderived from cancer patients with metastatic diseases. From this study,it is estimated that there are 8 to 80 million viable cancer cells inthe circulation of a cancer patient which have the potential tometastasize. This is over 100-fold higher resolution than previousantibody-based approach (Racila, E., Euhus, D., Weiss, A. J., Rao, C.,McConnell, J., Terstappen, L. W., and Uhr, J. W., 1998, Detection andCharacterization of Carcinoma Cells in the Blood. Proceedings of theNational Academy of Sciences of the United States of America 95,4589-4594). However, the number of metastatic cells estimated from thisstudy represents only 0.1% of total circulating cancer cells as reportedin a previous investigation (Glaves et al., 1988, Br. J. Cancer57:32-35).

7. EXAMPLE Regulation of Fibroblast Migration on a Collagenous Matrix isDependenton a Novel Cell Surface Protease Complex

The data provided below, demonstrates that the formation of a novelprotease complex, consisting of serine integral membrane proteases(SIMP), including seprase and dipeptidgl peptidase IV (DPPIV), atinvadopodia of migrating fibroblasts is associated with cell invasionand migration on a collagenous matrix.

7.1 Materials and Methods 7.1.1 Cell Culture

The human embryonic lung fibroblastic line WI38 and human breastcarcinoma cell line MDA-MB-436 were obtained from American Type CultureCollection (Rockville, Md.). Cells were cultured in a 1:1 mixture ofDulbecco's modified Eagle's medium (DMEM) and RPMI1640 supplemented with10% calf serum, 5% Nu-serum (Collaborative Research, Inc., Bedford,Mass.), 2 mM L-glutamine, 1 unit/ml penicillin, 10 g/ml streptomycin,0.1 mM non-essential amino acids and 1 mM sodium pyruvate. Rat mAbs E26,E19 and F4 are directed against human placental DPPIV, rat mAbs D8 andD28 are against human placental seprase (Goldstein L A et al., 1997,Biochem. Biophys. Acta. 1361:11-19; Pinereiro-Sanchez, M L et al., 1997,J. Biol. Chem. 272: 7595-7601; correction (1998) J. Biol. Chem.272:13366), mAb C27 is directed against human melanoma β1 integrin andrat mAb C37 is directed against cell surface glycoprotein gp-90(Meuller, S C., et al., 1999, J. Biol. Chem. 274:24947-24952). Mouseanti-αv integrin and anti-β3 integrin mAbs were from American TypeCulture Collection (clone L230, catalog number HB8448 and clone AP-3,catalog number HB242, respectively).

7.1.2 Isolation of Seprase-DPPIV Complex

WI38 cells were seeded onto hydrated type I collagen films (rat tailtype I collagen at 0.2 mg/ml, Collaborative Biomedical Products, Bectonand Dickinson Labware, Bedford, Mass.) and cultured until 90%confluence. Surface biotinylation of WI38 monolayers were performedusing Suffo-NSH-Biotin (Pierce, Rockford, Ill.) according tomanufacturer's instructions. To harvest lysates, each culture plate waswashed 3 times with PBS and extracted with 125 1/cm² of RIPA solution(1.25% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in 50 mM Trisbuffer, pH 7.5). Extraction was performed for 2 h at 25° C., 25 rpm on arotary shaker (Bellco Orbital Shaker, Vineland, N.J.). The cell layerand buffer were removed and transferred to a 50 ml conical tube andincubated a further 3 h at 4° C. with end-over-end agitation. Theextract was clarified by centrifugation at 10,000×g for 20 min at 4° C.and the supernatants used for immunoprecipitation reactions.

To prepare immunoaffinity matrix, purified rat mAbs against membraneproteins (2.5 mg) were coupled to 1-ml CNBr-Sepharose 4 MB (PharmacalBiotech Inc., Piscataway, N.J.). 0.25-ml mAb-beads were used toimmunoprecipitate complexes from 25-ml cell extract overnight at 4° C.with end-over-end agitation. After 3 washes in 25 ml of extractionbuffer, the beads with coupled antibody-antigen complexes wereresuspended in 0.1% glycine-HCl (pH 2.4) buffer (equal to the beadvolume) and the sample was incubated for 5 min at 4° C. Immediately, thesample was transferred to an Amicon filter insert (0.45 m 400 lcapacity) and centrifuged 10 min at 10,000 rpm in an Eppendorf microfugeat 4° C. The bead filtrate was neutralized by addition of 2M Trizmabase. To determine the subunit composition of isolated proteincomplexes, immunoprecipitates of the surface-biotinylated complexes wereanalyzed for their protein profiles by SDS-PAGE, transferred tonitrocellulose membranes and detected with HPR-streptavidin (Sigma, St.Louis, Mo.) and the ECL system (Amersham, St. Louis, Mo.). Isolatedprotein complexes were also used for immunoblotting (with anti-seprase,-DPPIV, -β1 and -β3 integrin mAbs), for gelatin zymography (to detectseprase gelatinase activity), and for DPPIV substrate membrane overlayas described (Pinereiro-Sanchez, M L et al., 1997, J. Biol. Chem. 272:7595-7601; correction (1998) J. Biol. Chem. 272:13366). To test forcomplete elution of proteins from the beads, Laemmli sample buffer(equal to the bead volume) was added and samples were heated bymicrowaves (2 cycles on low setting 30 sec each, followed by 1 cycle onmedium for 30 sec). Then, the samples were immediately centrifuged at 4°C. The filtrates were subjected to assays described above.

7.1.3 Labeling of Collagen Fibers

Collagen was polymerized prior to biotin, fluorescein or rhodaminelabeling so that sites of polymerization were not perturbed. Labeledcollagen fibers were then solubilized in acidified water (pH 2.0), butcould be readily polymerized back to collagen fibers under experimentalconditions. Specifically, 10 ml type I collagen solution (rat-tail typeI collagen, 4.66 mg/ml, Collaborative Biomedical Products, Becton andDickinson Labware, Bedford, Mass.) was mixed with 10-ml DMEM at 4° C.The mixture was incubated for 30 min at 37° C. to allow polymerizationof the collagen fibers (gel). The gel was washed with 30-ml of couplingborate buffer, pH 9.3 (Sigma) for 30 min and then incubated with 30-mlborate buffer containing 3 mg of Sulfo-NSH-Biotin (Pierce), FluoresceinIsothiocyanate I hydrochloride (FITC) or Tetramethyl RhodamineIsothiocyanate (TRITC) (Research Organics Inc, Cleveland, Ohio) at 25°C. on a shaker. Conjugation was stopped by washing 3 times with PBS,followed by a 50-ml PBS washing for 2 days and a 50-ml distilled waterwash for another 2 days. Labeled collagen fibers were solubilized inacidic water (0.02N acetic acid) to a final concentration of 1 mg/ml.Labeled collagen monomers were mixed with equal volume of DMEM orp-buffer (300 mM NaCl in 50 mM ammonium bicarbonate buffer, pH 8.4) andincubated for 30 min at 37° C. to allow gel formation.

7.1.4 Cell Migration and Fluorescent Collagen Degradation Assays

Fluorescent collagen fibers overlaying a monolayer wound culture wereused to examine cell migration in collagen gel during wound closure.WI38 cells were grown in 2-well chambered cover slips (Lab-Tek,Rochester, N.Y.) to confluence. The monolayer was scratched with apipette tip to generate wound edges. Culture media were then replacedwith TRITC-collagen in DMEM (600 g/ml; 50 l/well) and the cultureallowed to gel in a CO₂ incubator for 30 min at 37° C. Media containingserum or inhibitory mAbs (300 l/well) were then added and their effectson cell migration and collagen degradation in real time were observedusing phase contrast and fluorescence microscopy (Nikon InvertedMicroscope). Cell migration and collagen degradation were quantified bymeasuring the areas of cell migration and fluorescent collagen removalby migratory cells using NIH Image 1.62b4/fat analysis program.

A microtiter version of the above assay was developed to measurecollagen degradation by migratory cells. In a 96-well tissue cultureplate (Nunc, Rochester, N.Y.), 50 l/well of TRITC-collagen solution (600g/ml) was first loaded and the solution allowed to gel in 37° C.incubator for 30 min. The TRITC-collagen gel was then overlaid with 50l/well of the TRITC-collagen solution containing 2×10⁵ cells and mAbs(50 g/ml), and the culture allowed to gel in a CO₂ incubator for 30 minat 37° C. The culture was then supplemented with 150 l of fresh mediaper well. All media were prepared free of phenol red. At times 100 l ofculture media from each well was removed to measure the release ofTRITC-collagen peptides using a fluorescent microplate reader withexcitation at 544 nm and emission at 590 nm (Molecular Devices fMax:Fluorescence Microplate Reader). Leucine incorporation was used todetermine metabolic activities of cells in culture conditions, in which150 l/well of media containing 2 Ci/ml ³H-Leucine was added into theculture, and the cell-collagen layers were solubilized in 5 ml ofscintillation fluid and counted in a scintillation counter (BeckmanLS-7500).

Stable transfectants of the breast carcinoma line MDA-MB-436 thatexpresses constitutively seprase and DPPIV were obtained. Plasmid pA11(pCR3.1 vector alone), pA15 (vector plus full length seprase), and pZ8(DPPIV ribozyme construct) were transfected into MDA-MB-436 cells usinglipofectamine (Gibco/BRL) following the manufacture's instructions. Theselection medium contained G418 at a concentration of 300 g/ml.

7.1.5 Immunofluorescent Labeling of Seprase and DPPIV

WI38 cells were cultured in collagen gel, fixed and immunolabeled in asingle step using rhodamine conjugated mAbD28 against seprase andfluorescence conjugated mAb E26 against DPPIV as described (Meuller, SC, et al., 1999, J. Biol. Chem. 274:24947-24952). Stained samples werephotographed using the Planapo 25/1.2 or 63/1.4 objectives on a ZeissPhotomicroscope III (Carl Zeiss, Inc.) under epifluorescence.

7.1.6 Human Gingival Wounds and Invasive Human Breast Carcinoma

Human gingival biopsies were derived from the University of Turku,Finland. Full thickness wounds of oral mucosa were made from two healthyvolunteers and biopsies were collected after 3, 7, 14 and 28 days ofwounding. Immediately after biopsy, fresh tissue blocks were mounted inHistoprep® (Fisher Scientific, New Jersey) and snap frozen in liquidnitrogen. Frozen sections (6 m) were cut, fixed with −20° C. acetone for5 min, and stored at −70° C. For routine histology, the sections werestained with hematoxylin and eosin. For immunohistochemical staining,sections were washed with PBS containing 0.1% bovine serum albumin (BSA;Sigma Chemical Co., St. Louis, Mo.) and incubated withrhodamine-conjugated mAb D28 against seprase in PBS/BSA in humid chamberat 4° C. for 16 h. The sections were then washed with PBS/BSA and water,briefly air-dried, and mounted using cyanoacrylate glue (Krazy Glue,Borden Company LTD). The staining was examined using a Zeiss Axioskop 20light, fluorescence and confocal microscopy, and photographed using MC80 Zeiss microscope camera. Control staining was performed withrhodamine-conjugated secondary antibody and showed no specific stain.Immunohistochemical staining of invasive human breast carcinoma wasperformed as described (Kelly T et al., 1998, Mod. Pathol. 11:855-863).

7.2 Results

The data presented indicates that like seprase in LOX human malignantmelanoma cells (Pineiro-Snachez M L, 1997, J. Biol. Chem.272:7595-7601), the majority of seprase and DPPIV in WI38 humanembryonic fibroblasts were present as a >400-kDa complex in non-ionicdetergents, including Triton X-100, Triton X-114, RIPA buffer containing0.1% sodium dodecyl sulfate (SDS), and octyl glucoside, and inWGA-agarose affinity-purified material. The >400-kDa complex eluted inthe void volume fractions on Sephacryl S-200 gel filtrationchromatography. Isolation of WGA-purified material followed by Superose12 gel filtration liquid chromatography, exhibited major forms of about200-kDa (Fraction 17), 440-kDA (Fraction 14), and 670-kDa (Fraction 13)(FIG. 11A). As seprase contains a 97-kDa subunit and DPPIV a 110-kDamonomer and as both seprase and DPPIV are dimers in non-ionic detergents(10), the gel filtration data suggests the presence of the seprase-DPPIVcomplex at 440-670 kDa sizes (FIG. 11B).

Immunoprecipitation using mAb D28 (against seprase) and mAb E19 (againstDPPIV) identified two major similar intensity bands in the WI38 cellextract that was surface-biotinylated (FIG. 12A). The two bands,cross-immunoprecipitated by mAb D28 or E19, indicate seprase and DPPIVdimers, respectively. In such SDS gels when samples were solubilized in1% SDS and not boiled, the top or slower band at 200-kDa was identifiedby immunoblotting as DPPIV, and the lower or faster band migrating at170-kDa as seprase, respectively (FIG. 12B). The 350-400 kDa heteromericaggregate was not detected in SDS gels following SDS solubilization ofthe sample (FIGS. 12A, 12B), suggesting that the heteromeric aggregatedissociated into two stable dimers of 200-kDa DPPIV and 170-kDa seprase,respectively. In three independent experiments involving RIPA cellextracts, a stable association of seprase and DPPIV was detected usingmAbs against seprase and DPPIV but not those against β1 and β3 integrins(FIGS. 12A, 12B). Furthermore, such heteromeric complex was demonstratedby the proteolytic activities of the immuno-isolated complex. Antigenswere isolated from WI38 RIPA extracts by affinity purification usingeither mAb D28 or mAb E19 that recognize seprase or DPPIV, respectively.The eluates were analyzed for a 170-kDa (seprase) gelatinase (FIG. 12C)and 200-kDa (DPPIV) proline-specific dipeptidyl-aminopeptidase (FIG.12D). Gelatin zymography detected a 170-kDa gelatinase activity inimmunoprecipitates of anti-seprase mAb D28 (FIG. 12C, IP: seprase) oranti-DPPIV mAb E19 (FIG. 12C, IP: DPPIV). Isolated DPPIV dimer has nogelatinase activity and the 170-kDa band on the gelatin zymogramidentified with the DPPIV antibody represents the presence of seprase inthe complex. Similarly, DPPIV substrate overlay assay detected a 200-kDaproline-specific dipeptidyl-aminopeptidase activity inimmunoprecipitates of anti-seprase mAb D28 (FIG. 12D, IP: seprase) oranti-DPPIV mAb E19 (FIG. 12D, IP: DPPIV). No 170-kDa gelatinase of DPPIVactivities could be observed for αv, α2, α6 or β3 integrin or controlimmunoprecipitates. These results also confirm previous observationsthat seprase and DPPIV are homodimers in SDS buffer, which are sensitiveto heat (>60° C.) and acid for dissociation into their monomericsubunits. Thus it appears that the protease complex contained equalamounts of seprase and DPPIV proteins (FIGS. 11A, 11B, 12A, 12B), and itwas equally effective in eliciting 170-kDa gelatinase and 200-kDa DPPIVdieptidyl-aminopeptidase activities (FIGS. 12C, 12D).

To determine the role of the seprase-DPPIV complex in cell migration incollagen gel and possible collagen degradation we overlaid a thin layerof collagen on a cell monolayer wound model for morphologicalexamination and on a sparse culture for biochemical study (FIG. 13).Cell migration in collagen gel and local collagen removal by cells weremeasured by counting the area of cell migration/collagen removal usingimage analysis in conjunction with phase contrast and fluorescencemicroscopy (FIGS. 13A-13D). Addition of mAb E19 (against DPPIV) into thewound-closure model blocked cell migration (FIGS. 13B, 13C) and localcollagen removal by cells (FIG. 13D), while that of a class matched mAb(IgG) did not (FIGS. 13B, 13C, 13D). There was an increase in inhibitionwith increasing amounts of mAb E19 (FIG. 13B) and the antibodyinhibitory effect could be reversed by removal of E19, thus, this mAbwas not toxic (FIG. 13C). Furthermore, local collagen degradation byactivated cells was quantified by counting fluorescent peptides releasedfrom fluorescent collagen fibers by WI38 cells in a sparse culture in a96-well plate using spectrofluorometry (FIG. 13E). Cells in sparseculture are known to be migratory due to less “contact inhibition ofmigration” (Chen, W-T, 1979, J. Cell Biol 81:684-691). Migratory WI38cells showed time-dependent collagen degradation within 4 days, and mAbE19 (against DPPIV) inhibited collagen degradation by migratory cellswhile the control mAb C37 (anti-gp-90) did not (FIG. 13E). These datademonstrate the role of the seprase-DPPIV complex in cellular migrationin collagen gel and in the collagen degradation by wound-activatedfibroblasts.

As DPPIV was shown to be an adhesion receptor for collagen (Bauvois B,1988, Biochem. J. 252:723-731; Hanski C, 1988, Exp. Cell Res. 178:64-72;Loster K., 1995, Biochem. Biophys. Res. Commun. 217:341-348) orfibronectin (Cheng, H C 1998, J. Biol. Chem. 272:24207-24215; Johnson RC et al., 1993, J. Cell Biol 121:1423-1432; Piazza, G A et al., 1989,Biochem. J. 262:327-334)), it was determined whether the inhibitoryeffect of mAb E19 (against DPPIV) on cellular migration in collagen geland on the collagen degradation by migratory cells is due to itsinfluence on adhesion activity. FIG. 14 shows that, in parallelcomparison to integrin adhesion to collagen fibers, while mAb E19(against DPPIV) inhibits cellular migration in collagen gel and on thecollagen degradation by migratory cells it does not affect WI38 cellspreading on collagen substratum (FIG. 14A) and attachment to collagensubstratum (FIG. 14B). However, mAb C27 (against β1 integrins) inhibitsWI38 cell spreading on and adhesion to collagen substratum but mAb E19(against DPPIV) or mAb C37 (anti-gp-90) do not (FIGS. 14A and 14B).These indicate that β1 integrins may be primary collagen receptors onWI38 cells responsible for substrate binding of the seprase-DPPIVcomplex.

To demonstrate that seprase and DPPIV are associated in invadapodia ofmigratory cells, double label, immunofluorescence experiments wereperformed (FIG. 15). It was observed that invadapodia of the cellmigrating in collagen gel (a) were stained positively with TRITC-mAb D28against seprase (b) or FITC-mAB E26 against DPPIV (c). Superimposedimage also shows that seprase and DDPIV co-localize at the invadapodiaof a WI38 fibroblast migrating in collagen gel (d).

To examine if such seprase and DPPIV co-localization could be found invivo immunohistochemical experiments were performed on serial sectionsof formaldehyde-fixed, paraffin-embedded, human breast carcinoma tissue(FIGS. 16 and 18). Like tumor cells in the invasion front, connectivetissue cells in human invasive breast carcinoma were strongly reactivewith mAb D28 against seprase or mAB E26 against DDPIV (FIG. 16, arrows).However, such seprase and DPPIV staining was not detected in connectivetissue cells of distant normal tissues (FIG. 18). It is likely thatseprase-DDPIV expression associates with the activation of connectivetissue cells in response to cancer invasion; and seprase and DPPIVco-localize in these activated tissue cells as well as in invasivecarcinoma cells.

Unlike human umbilical cord smooth muscle cells in culture (Goldstein LA et al., 1997, Biochem. Biophys. Acta. 1361:11-19), both seprase andDPPIV preferentially distribute among mesenchymal cells but notdifferentiated muscle and endothelial cells of large vessels in humanembryonic tissues, including placenta and umbilical cord. To determineif seprase and DDPIV expression in stromal fibrobalsts is induced duringwound closure in vivo, the immunohistochemistry of human gingicvalmucosa-wound closure was investigated (FIG. 17). A strong expression ofseprase and DDPIV was seen in connective tissue cells at day 3 afterwounding (b-d, g). No immuno-reaction was seen in adjacent normal mucosatissue. No specific reaction was seen in the fibrin clot area andepithelium. Confocal microscopy shows the localization of seprase (d)and DPPIV in protrusions of connective tissue cells, which indicate theexistence of invadapodia in vivo. Later, at day 7 after wounding, only afew cells in the middle of granulation tissue were reactive with theanti-seprase antibody (f) but not the anti-DPPIV antibody (h). Sepraseand DDPIV staining disappeared from connective tissue cells after oneweek and cells of 14 or 28-day-old wounds also did not react with theantibody. The data indicates that seprase and DDPIV are activationenzymes on fibroblastic, endothelial and carcinoma cells, and that theymay participate in the local collagen degradation necessary for cellularmigration.

Cell transfection experiments were used to explore the cell surfaceassociation of seprase and DPPIV. MDA-MB-436 breast carcinoma cells thatexpress constitutively the seprase-DPPIV complex were used. Cellsover-expressing seprase or defective in DPPIV or seprase production wereexamined for their collagen-degrading and migratory activities incollagen gels (FIG. 19). An increase in release of TRITC-collagenpeptides was observed in MDA-MB-436 cells transfected with plasmid pA15that encodes seprase (FIG. 19A, pA15) and a decrease of peptide releasewas seen in cells transfected with a construct that encodes a DPPIVspecific ribozyme (FIG. 19A, pZ8) as compared to parental or vectortransfected cells (FIG. 19A; Parent, pA11). Also, over-expression ofseprase appears to associate with a slight reduction of DPPIV, and cellstransfected with the DPPIV ribozyme produce no detectable DPPIV andsubstantially reduced seprase (FIG. 19B).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeedvarious modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims. Variouspublications are cited herein that are hereby incorporated by referencein their entireties.

1. A method for determining the metastatic potential of cells derivedfrom a subject comprising: (a) isolating cancer cells from a blood fluidof the subject, said fluid selected from the group consisting of wholeblood, lymph, bone marrow, ascites or a combination thereof, said fluidcontaining a mixed population of cancer and non-cancer cells; saidisolating step comprising contacting said fluid with a cell adhesionmatrix for a time sufficient to allow the cancer cells in the fluid toselectively adhere to the matrix, said matrix comprising a fibrousscaffold comprising type I collagen fibers, type III collagen fibers,fibrin, cotton fibers, tissue fragment, or plastic fibers; wherein saidmatrix is coated with adhesion molecules, and wherein said isolatingstep has a level of sensitivity of one viable cancer cell per ml ofblood fluid; (b) incubating the isolated cancer cells for a timesufficient to allow ingestion of the matrix by the cells to occur; (c)detecting the ingestion of the matrix by the isolated cancer cellswherein a detection of cell ingestion of the matrix is an indicator ofcells with metastatic potential.
 2. The method of claim 1, wherein thematrix is labeled with a fluorescent label.
 3. The method of claim 1,wherein the matrix is labeled with a radioactive label.
 4. The method ofclaim 1, wherein the matrix is labeled with a color label.
 5. The methodof claim 1, wherein the matrix is labeled with a cytotoxic compound. 6.The method of claim 1, wherein the adhesion molecules comprisefibronectin.
 7. The method of claim 1, wherein the adhesion moleculescomprise laminin.
 8. The method of claim 1, wherein the adhesionmolecules comprise vitronectin.